Antibodies specific for claudin 6 (CLDN6)

ABSTRACT

The present invention provides antibodies useful as therapeutics for treating and/or preventing diseases associated with cells expressing Claudin-6 (CLDN6), including tumor-related diseases such as ovarian cancer, lung cancer, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, cancer of the urinary bladder, kidney cancer, colon cancer, placental choriocarcinoma, cervical cancer, testicular cancer, and uterine cancer.

This application is a National Stage Entry of PCT/EP2010/006888, whichwas filed on Nov. 11, 2010 and claimed priority to European PatentApplication Number 09014136.7 and U.S. Patent Application Ser. No.61/260,202, which were filed on Nov. 11, 2009 and European PatentApplication Number 10006956.6 and U.S. Patent Application Ser. No.61/361,618, which were filed on Jul. 6, 2010. The contents of each ofthe aforementioned applications are incorporated herein by reference intheir entireties.

Antibodies have been successfully introduced into the clinic for use incancer therapy and have emerged as the most promising therapeutics inoncology over the last decade. Antibody-based therapies for cancer havethe potential of higher specificity and lower side effect profile ascompared to conventional drugs. The reason is a precise distinctionbetween normal and neoplastic cells by antibodies and the fact thattheir mode of action relies on less toxic immunological anti-tumormechanisms, such as complement activation and recruitment of cytotoxicimmune cells.

Claudins are integral membrane proteins located within the tightjunctions of epithelia and endothelia. Claudins are predicted to havefour transmembrane segments with two extracellular loops, and N and Ctermini located in the cytoplasm. The claudin (CLDN) family oftransmembrane proteins plays a critical role in the maintenance ofepithelial and endothelial tight junctions and might also play a role inthe maintenance of the cytoskeleton and in cell signaling. Thedifferential expression of these proteins between tumor and normalcells, in addition to their membrane localization, makes them attractivetargets for cancer immunotherapy and the use of antibody-basedtherapeutics for targeting CLDNs in cancer therapy promises a high levelof therapeutic specificity.

However, the clinical application of CLDN-targeted therapeutics facesseveral obstacles. The ubiquitous expression of CLDNs in the body andthe critical role of CLDNs in the maintenance of tight junctionsrequires target specificity of CLDN-targeted therapeutics in order tomaximize treatment specificity and minimize systemic toxicity.

WO 2009/087978 relates to anti-CLDN6 antibodies and to their use asanti-cancer agents. In particular, the monoclonal antibodies designatedAB3-1, AE1-16, AE49-11, and AE3-20 are described. However, none of theseantibodies was specific for CLDN6 as shown by FACS analysis in Example5. Antibody AE3-20 reacted with CLDN9, while the antibodies AE1-16 andAE49-11 showed considerable reactivity with CLDN9 and also reacted withCLDN4. The binding of antibody AB3-1 to CLDN6 was as strong as itsbinding to CLDN9. It is described in Example 7 that the antibody AE49-11when administered to a mouse tumor model tended to inhibit tumor growthand had a life-prolonging effect. However, given the unspecificity ofthe antibody used, it remains unclear whether the described effects aredue to binding of the antibody to CLDN6.

Thus, up to now, no CLDN6-specific antibody has been described thatselectively binds to the surface of cells expressing CLDN6. However,such specific antibody would be required for antibody-based therapeuticapproaches using CLDN6 as a target.

The sequence alignment of CLDN3, CLDN4, CLDN6 and CLDN9 shown in FIG. 1illustrates that there is a high degree of conservation of CLDN6 toother claudin proteins. This high homology of CLDN6 with other claudinproteins, in particular CLDN9 and CLDN4, and the fact that WO2009/087978 failed to provide CLDN6-specific antibodies suggest that itmight not be possible to produce antibodies specifically binding toCLDN6.

SUMMARY OF THE INVENTION

The experimental results disclosed herein confirm that CLDN6 isexpressed in different human cancer cells while expression in normaltissues is limited to placenta.

Furthermore, the present invention for the first time describes thesuccessful production of CLDN6-specific antibodies capable of binding tothe surface of intact cells that express CLDN6. FACS analyzes of intactcells expressing CLDN6 showed the specific binding of anti-CLDN6antibodies while no binding was observed for cells expressing otherclaudin proteins, in particular, CLDN3, CLDN4 and CLDN9, or cells notexpressing any of these CLDN proteins. Thus, the present inventionunexpectedly demonstrates that an antibody can be produced specificallyperforming an antigen-antibody reaction with CLDN6 on the surface ofcells expressing CLDN6, but not substantially performing theantigen-antibody reaction with other highly homologous claudins.

The present invention generally provides antibodies useful astherapeutics for treating and/or preventing diseases associated withcells expressing CLDN6 and being characterized by association of CLDN6with their cell surface, including tumor-related diseases, in particularcancer, such as ovarian cancer, in particular ovarian adenocarcinoma andovarian teratocarcinoma, lung cancer, including small cell lung cancer(SCLC) and non-small cell lung cancer (NSCLC), in particular squamouscell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer,hepatic cancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer, agerm cell tumor such as a teratocarcinoma or an embryonal carcinoma, inparticular a germ cell tumor of the testis, and the metastatic formsthereof.

In one aspect the invention relates to an antibody which is capable ofbinding to CLDN6 associated with the surface of a cell that expressesCLDN6. Preferably, the antibody is not substantially capable of bindingto CLDN9 associated with the surface of a cell that expresses CLDN9.Preferably, the antibody is not substantially capable of binding toCLDN4 associated with the surface of a cell that expresses CLDN4 and/oris not substantially capable of binding to CLDN3 associated with thesurface of a cell that expresses CLDN3. Most preferably, the antibody isnot substantially capable of binding to a CLDN protein other than CLDN6associated with the surface of a cell that expresses said CLDN proteinand is specific for CLDN6. Preferably, said cell expressing said CLDNprotein is an intact cell, in particular a non-permeabilized cell, andsaid CLDN protein associated with the surface of a cell has a native,i.e. non-denatured, conformation. Preferably, the antibody is capable ofbinding to one or more epitopes of CLDN6 in their native conformation.

In one embodiment, the antibody is capable of binding to an epitopelocated within an extracellular portion of CLDN6, wherein saidextracellular portion of CLDN6 preferably comprises the amino acidsequence of any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQID NO: 15, preferably the amino acid sequence of SEQ ID NO: 6 or SEQ IDNO: 7, more preferably the amino acid sequence of SEQ ID NO: 6.Preferably, the antibody is capable of binding to an epitope locatedwithin the amino acid sequence of any one of SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 14 and SEQ ID NO: 15, preferably the amino acid sequence ofSEQ ID NO: 6 or SEQ ID NO: 7.

In one embodiment, the antibody is capable of binding to CLDN6 byinteracting at least with one, preferably more than one, such as 2, 3, 4or 5, preferably all amino acids selected from the group consisting ofThr33, Phe35, Gly37, Ser39, Ile40 and Leu151, preferably by interactingat least with one, preferably more than one, preferably all amino acidsselected from the group consisting of Thr33, Phe35, Gly37, Ser39 andIle40, more preferably by interacting at least with one, preferably morethan one, preferably all amino acids selected from the group consistingof Phe35, Gly37, Ser39 and Ile40 or consisting of Thr33, Phe35, Gly37and Ser39, and, in particular, by interacting at least with one,preferably more than one, preferably all amino acids selected from thegroup consisting of Phe35, Gly37 and Ser39. Preferably, the antibodydoes not interact with one or more, preferably all amino acids selectedfrom the group consisting of Glu154, Ala155, Arg158 and Gly161, andpreferably does not interact with one or more, preferably all aminoacids selected from the group consisting of Arg158 and Gly161.

The interaction between an antibody and CLDN6, in particular in itsnative conformation, can be analyzed by an alanine scanning mutagenesisof amino acids. CLDN6 mutants can be assessed for their ability to bebound by specific monoclonal antibodies. Impaired binding of a specificmonoclonal antibody to a CLDN6 mutant suggest that the mutated aminoacid is an important contact residue. Binding can be analyzed, forexample, by flow cytometry.

In one embodiment, the antibody is obtainable by a method comprising thestep of immunizing an animal with a peptide having the amino acidsequence of any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQID NO: 15, preferably the amino acid sequence of SEQ ID NO: 6 or SEQ IDNO: 7 or an immunologically equivalent peptide, or a nucleic acid orhost cell expressing said peptide.

In different embodiments, the CLDN6 to which the antibody is capable ofbinding has the amino acid sequence of SEQ ID NO: 2 or the amino acidsequence of SEQ ID NO: 8. It is particularly preferred that the antibodyis capable of binding to CLDN6 having the amino acid sequence of SEQ IDNO: 2 and capable of binding to CLDN6 having the amino acid sequence ofSEQ ID NO: 8.

In one embodiment, an antibody of the invention comprises an antibodyheavy chain comprising at least one, preferably two, more preferably allthree of the CDR sequences of an antibody heavy chain sequence selectedfrom SEQ ID NOs: 34, 36, 38 and 40, or a variant thereof. The CDRsequences are marked by a box in the above mentioned antibody heavychain sequences given in FIG. 25.

In one embodiment, an antibody of the invention comprises an antibodyheavy chain comprising the CDR3 sequence Xaa1 Gly Xaa2 Val Xaa3, whereinXaa1 is any amino acid, preferably an aromatic amino acid, morepreferably Phe or Tyr, most preferably Tyr, Xaa2 is any amino acid,preferably an aromatic amino acid, more preferably Phe or Tyr, mostpreferably Tyr, and Xaa3 is any amino acid, preferably Leu or Phe, morepreferably Leu. In one embodiment, an antibody of the inventioncomprises an antibody heavy chain comprising the CDR3 sequence Asp Xaa1Gly Xaa2 Val Xaa3 or Xaa1 Gly Xaa2 Val Xaa3 Asp, wherein Xaa1, Xaa2 andXaa3 are as defined above. In one embodiment, an antibody of theinvention comprises an antibody heavy chain comprising the CDR3 sequenceAsp Xaa1 Gly Xaa2 Val Xaa3 Asp, wherein Xaa1, Xaa2, and Xaa3 are asdefined above. In one embodiment, an antibody of the invention comprisesan antibody heavy chain comprising the CDR3 sequence Ala Arg Asp Xaa1Gly Xaa2 Val Xaa3 Asp Tyr, wherein Xaa1, Xaa2 and Xaa3 are as definedabove. In one embodiment, an antibody according to the foregoingembodiments comprises an antibody heavy chain comprising the CDR1sequence according to SEQ ID NO: 47 or a variant thereof and/or the CDR2sequence according to SEQ ID NO: 48 or a variant thereof.

In one embodiment, an antibody of the invention comprises an antibodyheavy chain comprising an antibody heavy chain sequence selected fromSEQ ID NOs: 34, 36, 38 and 40 or a variant thereof.

In one embodiment, an antibody of the invention comprises an antibodylight chain comprising at least one, preferably two, more preferably allthree of the CDR sequences of an antibody light chain sequence selectedfrom SEQ ID NOs: 35, 37, 39 and 41, or a variant thereof. The CDRsequences are marked by a box in the above mentioned antibody lightchain sequences given in FIG. 26.

In one embodiment, an antibody of the invention comprises an antibodylight chain comprising the CDR3 sequence Arg Xaa1 Xaa2 Xaa3 Pro, whereinXaa1 is any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2is any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferablyTyr, Ser, or Asn, most preferably Asn, and Xaa3 is any amino acid,preferably Ser or Tyr, more preferably Tyr. In one embodiment, anantibody of the invention comprises an antibody light chain comprisingthe CDR3 sequence Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro, wherein Xaa1, Xaa2 andXaa3 are as defined above. In one embodiment, an antibody of theinvention comprises an antibody light chain comprising the CDR3 sequenceGln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, wherein Xaa1, Xaa2 and Xaa3are as defined above. In one embodiment, an antibody according to theforegoing embodiments comprises an antibody light chain comprising theCDR1 sequence according to SEQ ID NO: 52 or a variant thereof and/or theCDR2 sequence according to SEQ ID NO: 53 or a variant thereof.

In one embodiment, an antibody of the invention comprises an antibodylight chain comprising an antibody light chain sequence selected fromSEQ ID NOs: 35, 37, 39 and 41 or a variant thereof.

In various embodiments, an antibody of the invention comprises anantibody heavy chain as discussed above and an antibody light chain asalso discussed above.

In one embodiment, an antibody of the invention comprises:

(i) an antibody heavy chain comprising at least one, preferably two,more preferably all three of the CDR sequences of an antibody heavychain sequence of SEQ ID NO: x, or a variant thereof, and

(ii) an antibody light chain comprising at least one, preferably two,more preferably all three of the CDR sequences of an antibody lightchain sequence of SEQ ID NO: x+1, or a variant thereof;

wherein x selected from 34, 36, 38 and 40.

The CDR sequences are marked by a box in the above mentioned antibodyheavy chain sequences and antibody light chain sequences, respectively,given in FIG. 25 and FIG. 26, respectively.

In one embodiment, an antibody of the invention comprises:

(i) an antibody heavy chain comprising a CDR3 sequence selected from thegroup consisting of Xaa1 Gly Xaa2 Val Xaa3, Asp Xaa1 Gly Xaa2 Val Xaa3,Xaa1 Gly Xaa2 Val Xaa3 Asp, Asp Xaa1 Gly Xaa2 Val Xaa3 Asp, and Ala ArgAsp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, wherein Xaa1 is any amino acid,preferably an aromatic amino acid, more preferably Phe or Tyr, mostpreferably Tyr, Xaa2 is any amino acid, preferably an aromatic aminoacid, more preferably Phe or Tyr, most preferably Tyr, and Xaa3 is anyamino acid, preferably Leu or Phe, more preferably Leu, and(ii) an antibody light chain comprising a CDR3 sequence selected fromthe group consisting of Arg Xaa1 Xaa2 Xaa3 Pro, Gln Arg Xaa1 Xaa2 Xaa3Pro Pro, Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, wherein Xaa1 is anyamino acid, preferably Ser or Asn, most preferably Ser, Xaa2 is anyamino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferably Tyr,Ser, or Asn, most preferably Asn, and Xaa3 is any amino acid, preferablySer or Tyr, more preferably Tyr.

In one embodiment, an antibody according to the foregoing embodimentscomprises (i) an antibody heavy chain comprising the CDR1 sequenceaccording to SEQ ID NO: 47 or a variant thereof and/or the CDR2 sequenceaccording to SEQ ID NO: 48 or a variant thereof and/or (ii) an antibodylight chain comprising the CDR1 sequence according to SEQ ID NO: 52 or avariant thereof and/or the CDR2 sequence according to SEQ ID NO: 53 or avariant thereof.

In one embodiment, an antibody of the invention comprises:

(i) an antibody heavy chain comprising an antibody heavy chain sequenceof SEQ ID NO: x or a variant thereof, and

(ii) an antibody light chain comprising an antibody light chain sequenceof SEQ ID NO: x+1 or a variant thereof;

wherein x selected from 34, 36, 38 and 40.

In preferred embodiments, the antibody has one or more of the followingactivities: (i) killing of a cell expressing CLDN6, (ii) inhibition ofproliferation of a cell expressing CLDN6, (iii) inhibition of colonyformation of a cell expressing CLDN6, (iv) mediating remission, i.e.reduction in size, preferably complete remission, i.e. completedisappearance, of established tumors, (v) prevention of the formation orre-formation of tumors, and (vi) inhibition of metastasis of a cellexpressing CLDN6. Accordingly, the antibody may be used for one or moreof the foregoing, in particular when administered to a patient. Suchkilling of cells and/or inhibition of one or more activities of cellscan be utilized therapeutically as described herein. In particular,killing of cells, inhibition of proliferation of cells and/or inhibitionof colony formation of cells can be utilized for treating or preventingcancer, including cancer metastasis. Inhibition of proliferation, colonyformation and/or metastasis of cells can be utilized, in particular, fortreating or preventing cancer metastasis and the metastatic spread ofcancer cells. Preferably the antibody of the invention mediates killingof cells by inducing complement dependent cytotoxicity (CDC) mediatedlysis, antibody dependent cellular cytotoxicity (ADCC) mediated lysis,apoptosis, homotypic adhesion, and/or phagocytosis, preferably byinducing CDC mediated lysis and/or ADCC mediated lysis. However, thepresent invention also includes embodiments wherein the antibody exertsits activity as described herein such as killing of cells and/orinhibition of one or more activities of cells, e.g. cell proliferationand/or colony formation, without inducing complement dependentcytotoxicity (CDC) mediated lysis, antibody dependent cellularcytotoxicity (ADCC) mediated lysis, apoptosis, homotypic adhesion,and/or phagocytosis. For example, the antibody of the invention may alsoexert an effect simply by binding to CLDN6 on the cell surface, thus,e.g. blocking proliferation of the cells. In one embodiment the antibodyof the invention does not induce CDC mediated lysis of cells.

Preferably, ADCC mediated lysis of cells takes place in the presence ofeffector cells, which in particular embodiments are selected from thegroup consisting of monocytes, mononuclear cells, NK cells and PMNs, andphagocytosis is by macrophages.

The activity of inhibiting or reducing proliferation of cells expressingCLDN6, preferably cancer cells, can be measured in vitro by determiningproliferation of CLDN6-expressing cancer cells in an assay usingbromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a syntheticnucleoside which is an analogue of thymidine and can be incorporatedinto the newly synthesized DNA of replicating cells (during the S phaseof the cell cycle), substituting for thymidine during DNA replication.Detecting the incorporated chemical using, for example, antibodiesspecific for BrdU indicates cells that were actively replicating theirDNA.

The activity of inhibiting or reducing colony formation of cellsexpressing CLDN6, preferably cancer cells, can be measured in vitro in aclonogenic assay. A clonogenic assay is a microbiology technique forstudying the effectiveness of specific agents on the survival andproliferation of cells. It is frequently used in cancer researchlaboratories to determine the effect of drugs or radiation onproliferating tumor cells. The experiment involves three major steps:(i) applying a treatment to a sample of cells, in particular cancercells, (ii) plating the cells in a tissue culture vessel and (iii)allowing the cells to grow. The colonies produced are fixed, stained,and counted. Colony formation is of importance with respect to theformation of metastases if individual tumor cells colonize organs. Theinhibitory activity of the antibodies indicates their potential insuppressing the formation of metastases. Antibodies having the activityof inhibiting or reducing colony formation in a clonogenic assay areparticularly useful for treating or preventing metastasis and themetastatic spread of cancer cells, in particular of the cancer typesmentioned herein.

In preferred embodiments, the antibody exhibits one or more immuneeffector functions against a cell carrying CLDN6 in its nativeconformation, wherein the one or more immune effector functions arepreferably selected from the group consisting of complement dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), induction of apoptosis, and inhibition of proliferation,preferably the effector functions are ADCC and/or CDC.

Preferably said one or more activities or one or more immune effectorfunctions exhibited by said antibody are induced by binding of saidantibody to CLDN6, preferably to an epitope located within anextracellular portion of CLDN6, wherein said extracellular portion ofCLDN6 preferably comprises the amino acid sequence of any one of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15, preferably theamino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, more preferably theamino acid sequence of SEQ ID NO: 6.

According to the invention, a cell expressing CLDN6 is preferablycharacterized by association of CLDN6 with its cell surface. A cellexpressing CLDN6 or a cell carrying CLDN6 in its native conformationpreferably is a tumor cell, such as a cancer cell, preferably a cancercell from a cancer selected from the group consisting of ovarian cancer,in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lungcancer, including small cell lung cancer (SCLC) and non-small cell lungcancer (NSCLC), in particular squamous cell lung carcinoma andadenocarcinoma, gastric cancer, breast cancer, hepatic cancer,pancreatic cancer, skin cancer, in particular basal cell carcinoma andsquamous cell carcinoma, malignant melanoma, head and neck cancer, inparticular malignant pleomorphic adenoma, sarcoma, in particularsynovial sarcoma and carcinosarcoma, bile duct cancer, cancer of theurinary bladder, in particular transitional cell carcinoma and papillarycarcinoma, kidney cancer, in particular renal cell carcinoma includingclear cell renal cell carcinoma and papillary renal cell carcinoma,colon cancer, small bowel cancer, including cancer of the ileum, inparticular small bowel adenocarcinoma and adenocarcinoma of the ileum,testicular embryonal carcinoma, placental choriocarcinoma, cervicalcancer, testicular cancer, in particular testicular seminoma, testicularteratoma and embryonic testicular cancer, uterine cancer, a germ celltumor such as a teratocarcinoma or an embryonal carcinoma, in particulara germ cell tumor of the testis, and the metastatic forms thereof.

The antibody of the invention may be attached to one or more therapeuticeffector moieties, e.g., radiolabels, cytotoxins, therapeutic enzymes,agents that induce apoptosis, and the like in order to provide fortargeted cytotoxicity, i.e., killing of tumor cells.

In one embodiment the antibody of the invention (i) binds to cellsexpressing CLDN6 and being characterized by association of CLDN6 withtheir cell surface, and (ii) does not bind to cells not expressing CLDN6and not being characterized by association of CLDN6 with their cellsurface. The antibody of the invention preferably (i) mediates killingand/or inhibits proliferation of cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface, and (ii)does not mediate killing and/or do not inhibit proliferation of cellsnot expressing CLDN6 and not being characterized by association of CLDN6with their cell surface.

In particular preferred embodiments, the antibody of the invention bindsto native epitopes of CLDN6 present on the surface of living cells suchas those of SEQ ID NOs: 6 or 7. In further preferred embodiments, theantibody of the invention is specific for CLDN6-expressing cancer cellsand does not bind to cancer cells not expressing CLDN6.

Antibodies of the invention may be derived from different species,including but not limited to mouse, rat, rabbit, guinea pig and human.Antibodies of the invention also include chimeric molecules in which anantibody constant region derived from one species, preferably human, iscombined with the antigen binding site derived from another species.Moreover antibodies of the invention include humanized molecules inwhich the antigen binding sites of an antibody derived from a non-humanspecies are combined with constant and framework regions of humanorigin.

Antibodies of the invention include polyclonal and monoclonal antibodiesand include IgG2a (e.g. IgG2a, κ, λ), IgG2b (e.g. IgG2b, κ, λ), IgG3(e.g. IgG3, κ, λ) and IgM antibodies. However, other antibody isotypesare also encompassed by the invention, including IgG1, IgA1, IgA2,secretory IgA, IgD, and IgE antibodies. The antibodies can be wholeantibodies or antigen-binding fragments thereof including, for example,Fab, F(ab′)₂, Fv, single chain Fv fragments or bispecific antibodies.Furthermore, the antigen-binding fragments include binding-domainimmunoglobulin fusion proteins comprising (i) a binding domainpolypeptide (such as a heavy chain variable region or a light chainvariable region) that is fused to an immunoglobulin hinge regionpolypeptide, (ii) an immunoglobulin heavy chain CH2 constant regionfused to the hinge region, and (iii) an immunoglobulin heavy chain CH3constant region fused to the CH2 constant region. Such binding-domainimmunoglobulin fusion proteins are further disclosed in US2003/0118592and US 2003/0133939.

The antibody of the invention preferably is a monoclonal, chimeric,human or humanized antibody, or a fragment of an antibody. Antibodies ofthe invention include fully human antibodies. Such antibodies may beproduced in a non-human transgenic animal, e.g., a transgenic mouse,capable of producing multiple isotypes of human monoclonal antibodies toCLDN6 by undergoing V-D-J recombination and isotype switching. Suchtransgenic animal can also be a transgenic rabbit for producingpolyclonal antibodies such as disclosed in US 2003/0017534.

Antibodies of the present invention preferably dissociate from CLDN6with a dissociation equilibrium constant (KD) of approximately 1-100 nMor less. Preferably, antibodies of the invention do not cross-react withrelated cell-surface antigens and thus do not inhibit their function.

In preferred embodiments, antibodies of the present invention can becharacterized by one or more of the following properties:

-   a) specificity for CLDN6;-   b) a binding affinity to CLDN6 of about 100 nM or less, preferably,    about 5-10 nM or less and, more preferably, about 1-3 nM or less,-   c) the ability to induce CDC of cells which express CLDN6 and are    characterized by association of CLDN6 with their cell surface;-   d) the ability to inhibit the growth of cells which express CLDN6    and are characterized by association of CLDN6 with their cell    surface;-   e) the ability to induce apoptosis of cells which express CLDN6 and    are characterized by association of CLDN6 with their cell surface;-   f) the ability to induce homotypic adhesion of cells which express    CLDN6 and are characterized by association of CLDN6 with their cell    surface;-   g) the ability to induce ADCC of cells which express CLDN6 and are    characterized by association of CLDN6 with their cell surface in the    presence of effector cells;-   h) the ability to prolong survival of a subject having tumor cells    which express CLDN6 and are characterized by association of CLDN6    with their cell surface;-   i) the ability to deplete cells which express CLDN6 and are    characterized by association of CLDN6 with their cell surface;-   j) the ability to aggregate CLDN6 on the surface of living cells.

A preferred antibody described herein is an antibody produced by orobtainable from a hybridoma cell deposited at the DSMZ (Inhoffenstr. 7B,38124 Braunschweig, Germany) and having one of the followingdesignations and accession numbers:

1. GT512muMAB 59A, accession no. DSM ACC3067, deposited on Jun. 21,2010;

2. GT512muMAB 60A, accession no. DSM ACC3068, deposited on Jun. 21,2010;

3. GT512muMAB 61D, accession no. DSM ACC3069, deposited on Jun. 21,2010;

4. GT512muMAB 64A, accession no. DSM ACC3070, deposited on Jun. 21,2010;

5. GT512muMAB 65A, accession no. DSM ACC3071, deposited on Jun. 21,2010;

6. GT512muMAB 66B, accession no. DSM ACC3072, deposited on Jun. 21,2010;

7. GT512muMAB 67A, accession no. DSM ACC3073, deposited on Jun. 21,2010;

8. GT512muMAB 55A, accession no. DSM ACC3089, deposited on Aug. 31,2010; or

9. GT512muMAB 89A, accession no. DSM ACC3090, deposited on Aug. 31,2010.

Antibodies of the invention are designated herein by referring to thedesignation of the antibody and/or by referring to the clone producingthe antibody, e.g. muMAB 59A.

Further preferred antibodies are those having the specificity of theantibodies produced by and obtainable from the above-describedhybridomas and, in particular, those comprising an antigen bindingportion or antigen binding site, in particular a variable region,identical or highly homologous to that of the antibodies produced by andobtainable from the above-described hybridomas. It is contemplated thatpreferred antibodies are those having CDR regions either identical orhighly homologous to the regions of antibodies produced by andobtainable from the above-described hybridomas. By “highly homologous”it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1to 3 or 1 or 2 substitutions may be made in each CDR region.Particularly preferred antibodies are the chimerized and humanized formsof the antibodies produced by and obtainable from the above-describedhybridomas.

Thus, an antibody of the invention may be selected from the groupconsisting of (i) an antibody produced by or obtainable from a clonedeposited under the accession no. DSM ACC3067 (GT512muMAB 59A), DSMACC3068 (GT512muMAB 60A), DSM ACC3069 (GT512muMAB 61D), DSM ACC3070(GT512muMAB 64A), DSM ACC3071 (GT512muMAB 65A), DSM ACC3072 (GT512muMAB66B), DSM ACC3073 (GT512muMAB 67A), DSM ACC3089 (GT512muMAB 55A), or DSMACC3090 (GT512muMAB 89A), (ii) an antibody which is a chimerized orhumanized form of the antibody under (i), (iii) an antibody which hasthe specificity of the antibody under (i), and (iv) an antibodycomprising the antigen binding portion or antigen binding site of theantibody under (i). The antigen binding portion or antigen binding siteof the antibody under (i) may comprise the variable region of theantibody under (i).

The present invention also relates to a cell such as a hybridoma cellproducing an antibody as described herein.

Preferred hybridoma cells are those deposited at the DSMZ (Inhoffenstr.7B, 38124 Braunschweig, Germany) and having one of the followingdesignations and accession numbers:

1. GT512muMAB 59A, accession no. DSM ACC3067, deposited on Jun. 21,2010;

2. GT512muMAB 60A, accession no. DSM ACC3068, deposited on Jun. 21,2010;

3. GT512muMAB 61D, accession no. DSM ACC3069, deposited on Jun. 21,2010;

4. GT512muMAB 64A, accession no. DSM ACC3070, deposited on Jun. 21,2010;

5. GT512muMAB 65A, accession no. DSM ACC3071, deposited on Jun. 21,2010;

6. GT512muMAB 66B, accession no. DSM ACC3072, deposited on Jun. 21,2010;

7. GT512muMAB 67A, accession no. DSM ACC3073, deposited on Jun. 21,2010;

8. GT512muMAB 55A, accession no. DSM ACC3089, deposited on Aug. 31,2010; or

9. GT512muMAB 89A, accession no. DSM ACC3090, deposited on Aug. 31,2010.

The anti-CLDN6 antibodies of the present invention can be derivatized,linked to or co-expressed to other binding specificities. In aparticular embodiment, the invention provides a bispecific ormultispecific molecule comprising at least one first binding specificityfor CLDN6 (e.g., an anti-CLDN6 antibody or mimetic thereof), and asecond binding specificity for a effector cell, such as a bindingspecificity for an Fc receptor (e.g., a Fc-gamma receptor, such asFc-gamma RI, or any other Fc receptor) or a T cell receptor, e.g., CD3.

Accordingly, the present invention includes bispecific and multispecificmolecules that bind to both CLDN6 and to an Fc receptor or a T cellreceptor, e.g. CD3. Examples of Fc receptors are IgG receptor, Fc-gammareceptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII(CD16). Other Fc receptors, such as IgA receptors (e.g., FcαRI), alsocan be targeted. The Fc receptor is preferably located on the surface ofan effector cell, e.g., a monocyte, macrophage or an activatedmononuclear cell. In a preferred embodiment, the bispecific andmultispecific molecules bind to an Fc receptor at a site which isdistinct from the immunoglobulin Fc (e.g., IgG or IgA) binding site ofthe receptor. Therefore, the binding of the bispecific and multispecificmolecules is not blocked by physiological levels of immunoglobulins.

In yet another aspect, anti-CLDN6 antibodies of the invention arederivatized, linked to or co-expressed with another functional molecule,e.g., another peptide or protein (e.g., a Fab′ fragment). For example,an antibody of the invention can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (e.g.to produce a bispecific or a multispecific antibody), a cytotoxin,cellular ligand or antigen (e.g. to produce an immunoconjugate, such asan immunotoxin). An antibody of the present invention can be linked toother therapeutic moieties, e.g., a radioisotope, a small moleculeanti-cancer drug, a recombinant cytokine or chemokine. Accordingly, thepresent invention encompasses a large variety of antibody conjugates,bispecific and multispecific molecules, and fusion proteins, all ofwhich bind to CLDN6 expressing cells and/or to cells being characterizedby association of CLDN6 with their cell surface and which can be used totarget other molecules to such cells.

Generally, for the purposes of the present invention, all antibodyderivatives such as antibody conjugates, bispecific and multispecificmolecules, and fusion proteins described herein are encompassed by theterm “antibody”.

In a further aspect, the invention also envisions CLDN6-binding proteinsderived from non-immunoglobulin domains, in particular single-chainproteins. Such binding proteins and methods for their production aredescribed, for example, in Binz et al. (2005) Nature Biotechnology 23(10): 1257-1268, herein incorporated by reference. It is to beunderstood that the teaching given herein with respect to immunoglobulinor immunoglobulin derived binding molecules correspondingly also appliesto binding molecules derived from non-immunoglobulin domains. Inparticular, using such binding molecules derived from non-immunoglobulindomains it is possible to block CLDN6 of cells expressing said targetand being characterized by association of said target with their cellsurface and thus, to bring about therapeutic effects as disclosed hereinfor antibodies of the invention, in particular the inhibition of one ormore activities of tumor cells as disclosed herein such asproliferation. Although not mandatory, it is possible to confer effectorfunctions of antibodies to such non-immunoglobulin binding molecules bye.g. fusion to the Fc region of antibodies.

The present invention generally embraces the treatment and/or diagnosisof diseases, in particular tumor diseases, by targeting CLDN6 expressedby cells and being associated with the surface of cells. These methodsprovide for the selective detection and/or eradication of such cellsthereby minimizing adverse effects to normal cells not expressing CLDN6and not being characterized by association of CLDN6 with their cellsurface. Preferred diseases for a therapy or diagnosis are those inwhich cells expressing CLDN6 and being characterized by association ofCLDN6 with their cell surface are involved such as tumor diseases, inparticular cancer diseases such as those described herein.

In one aspect, the invention provides compositions, e.g., pharmaceuticaland diagnostic compositions/kits, comprising an antibody or acombination of antibodies of the invention. A pharmaceutical compositionof the invention may comprise a pharmaceutically acceptable carrier andmay optionally comprise one or more adjuvants, stabilizers etc. In aparticular embodiment, the composition includes a combination ofantibodies which bind to distinct epitopes or which possess distinctfunctional characteristics, such as inducing CDC and/or ADCC andinducing apoptosis. In this embodiment of the invention, antibodies maybe used in combination, e.g., as a pharmaceutical composition comprisingtwo or more anti-CLDN6 monoclonal antibodies. For example, anti-CLDN6antibodies having different but complementary activities can be combinedin a single therapy to achieve a desired therapeutic effect. In apreferred embodiment, the composition includes an anti-CLDN6 antibodythat mediates CDC combined with another anti-CLDN6 antibody that inducesapoptosis. In another embodiment, the composition includes an anti-CLDN6antibody that mediates highly effective killing of target cells in thepresence of effector cells, combined with another anti-CLDN6 antibodythat inhibits the growth of cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface.

The present invention also includes the simultaneous or sequentialadministration of two or more anti-CLDN6 antibodies of the invention,wherein preferably at least one of said antibodies is a chimericanti-CLDN6 antibody and at least one further antibody is a humananti-CLDN6 antibody, the antibodies binding to the same or differentepitopes of CLDN6. Preferably, a chimeric CLDN6 antibody of theinvention is administered first followed by the administration of ahuman anti-CLDN6 antibody of the invention, wherein the human anti-CLDN6antibody is preferably administered for an extended period of time, i.e.as maintenance therapy.

Antibodies, conjugates, bispecific/multispecific molecules andcompositions of the present invention can be used in a variety ofmethods for inhibiting growth of cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface and/orselectively killing cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface by contacting the cellswith an effective amount of the antibody, conjugate,bispecific/multispecific molecule or composition, such that the growthof the cell is inhibited and/or the cell is killed. In one embodiment,the method includes killing of the cell expressing CLDN6 and beingcharacterized by association of CLDN6 with its cell surface, optionallyin the presence of effector cells, for example, by CDC, apoptosis, ADCC,phagocytosis, or by a combination of two or more of these mechanisms.Cells expressing CLDN6 and being characterized by association of CLDN6with their cell surface which can be inhibited or killed using theantibodies of the invention include cancer cells.

Antibodies, conjugates, and bispecific/multispecific molecules andcompositions of the present invention can be used to treat and/orprevent a variety of diseases involving cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface byadministering the antibodies to patients suffering from such diseases.Exemplary diseases that can be treated (e.g., ameliorated) or preventedinclude, but are not limited to, tumorigenic diseases. Examples oftumorigenic diseases, which can be treated and/or prevented, includecancer diseases such as ovarian cancer, in particular ovarianadenocarcinoma and ovarian teratocarcinoma, lung cancer, including smallcell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), inparticular squamous cell lung carcinoma and adenocarcinoma, gastriccancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer,in particular basal cell carcinoma and squamous cell carcinoma,malignant melanoma, head and neck cancer, in particular malignantpleomorphic adenoma, sarcoma, in particular synovial sarcoma andcarcinosarcoma, bile duct cancer, cancer of the urinary bladder, inparticular transitional cell carcinoma and papillary carcinoma, kidneycancer, in particular renal cell carcinoma including clear cell renalcell carcinoma and papillary renal cell carcinoma, colon cancer, smallbowel cancer, including cancer of the ileum, in particular small boweladenocarcinoma and adenocarcinoma of the ileum, testicular embryonalcarcinoma, placental choriocarcinoma, cervical cancer, testicularcancer, in particular testicular seminoma, testicular teratoma andembryonic testicular cancer, uterine cancer, a germ cell tumor such as ateratocarcinoma or an embryonal carcinoma, in particular a germ celltumor of the testis, and the metastatic forms thereof.

In a further aspect the invention relates to a method of treating orpreventing a disease or disorder involving cells expressing CLDN6 andbeing characterized by association of CLDN6 with their cell surfacecomprising administering to a subject the antibody, conjugate,bispecific/multispecific molecule or composition of the invention.Preferably the disease or disorder is a tumor-related disease and inparticular embodiments is selected from the group consisting of ovariancancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer, agerm cell tumor such as a teratocarcinoma or an embryonal carcinoma, inparticular a germ cell tumor of the testis, and the metastatic formsthereof. CLDN6 is preferably expressed on the surface of said cells.

The invention may involve the use of the agents and compositionsdescribed herein for a prophylactic and/or therapeutic treatment oftumor diseases, i.e. for treating a patient having a tumor disease orbeing at risk of developing a tumor disease. In one aspect, theinvention provides methods for inhibiting tumor growth comprising theadministration of one or more of the agents and compositions describedherein.

Preferably, the agents and compositions described herein areadministered in a way such that the therapeutically active substance isnot delivered or not substantially delivered to a tissue or organwherein the cells when the tissue or organ is free of tumors expressCLDN6 and are characterized by association of CLDN6 with their cellsurface such as placenta tissue or placenta. To this end, the agents andcompositions described herein can be administered locally.

In one aspect, the invention provides an antibody as described hereinfor use in the methods of treatment described herein. In one embodiment,the invention provides a pharmaceutical composition as described hereinfor use in the methods of treatment described herein.

In a particular embodiment of the invention, the subject beingadministered the antibody is additionally treated with achemotherapeutic agent, radiation, or an agent that modulates, e.g.,enhances or inhibits, the expression or activity of an Fc receptor, e.g.an Fc-gamma receptor, such as a cytokine. Typical cytokines foradministration during treatment include granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Typicaltherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin, cisplatin, taxotere, 5-fluoruracil, methotrexat, gemzitabinand cyclophosphamide.

In yet another aspect, the invention relates to an immunization strategyto immunize non-human animals such as mice with human CLDN6 or a peptidefragment thereof to obtain antibodies. Preferred peptides forimmunization are those selected from the group consisting of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15, and immunologicallyequivalent peptides.

Wildtype as well as transgenic non-human animals can be immunized with apurified or enriched preparation of CLDN6 antigen or a peptide fragmentthereof and/or nucleic acids and/or cells expressing CLDN6 or a peptidefragment thereof. Preferably, the transgenic non-human animal is capableof producing multiple isotypes of human monoclonal antibodies to CLDN6(e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination andisotype switching. Isotype switching may occur by e.g., classical ornon-classical isotype switching.

Accordingly, in yet another aspect, the invention provides isolated Bcells from a non-human animal as described above. The isolated B cellscan then be immortalized by fusion to an immortalized cell to provide asource (e.g., a hybridoma) of antibodies of the invention. Suchhybridomas (i.e., which produce antibodies of the invention) are alsoincluded within the scope of the invention.

In a further aspect, the present invention relates to methods fordiagnosis, detection or monitoring of a tumor disease comprising thedetection of and/or determination of the quantity of CLDN6 or cellsexpressing CLDN6 and being characterized by association of CLDN6 withtheir cell surface in a biological sample isolated from a patient usingan antibody of the invention. The biological sample may be isolated froma patient having a tumor disease, being suspected of having or fallingill with a tumor disease or having a potential for a tumor disease.

In one embodiment of the method for diagnosis, detection or monitoringof a tumor disease according to the invention, a biological sampleand/or a control/reference sample is from a tissue or organcorresponding to the tissue or organ which is to be diagnosed, detectedor monitored with respect to affection by a tumor disease, e.g. thetumor disease which is to be diagnosed, detected or monitored is ovariancancer and the biological sample and/or control/reference sample isovarian tissue. Such tissues and organs are described herein, forexample, in connection with different tumor diseases and cancers.

In one embodiment of the methods for diagnosis, detection or monitoringof a tumor disease the biological sample is from a tissue or organwherein the cells when the tissue or organ is free of tumors do notsubstantially express CLDN6 and are not characterized by substantialassociation of CLDN6 with their cell surface. Preferably said tissue isa tissue other than placenta tissue.

Typically, the level of a target molecule in a biological sample iscompared to a reference level, wherein a deviation from said referencelevel is indicative of the presence and/or stage of a tumor disease in asubject. The reference level may be a level as determined in a controlsample (e.g., from a healthy tissue or subject) or a median level fromhealthy subjects. A “deviation” from said reference level designates anysignificant change, such as an increase or decrease by at least 10%,20%, or 30%, preferably by at least 40% or 50%, or even more.Preferably, the presence of CLDN6 or cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface in saidbiological sample or a quantity of CLDN6 or cells expressing CLDN6 andbeing characterized by association of CLDN6 with their cell surface inthe biological sample which is increased compared to a reference levelindicates the presence of a tumor disease.

Typically, the detection and/or determination of the quantity in themethods of the invention involves the use of labeled antibodies whichspecifically bind to a target molecule.

In a particular aspect, the invention relates to a method for detection,i.e. determining the position or site, of a tumor disease, e.g. aparticular tissue or organ, which comprises administering an antibody ofthe present invention which is coupled to a detectable label to apatient. Labelling of a tissue or organ in said patient may indicate thepresence of or risk for a tumor disease in said tissue or organ.

As exemplified herein, antibodies of the invention can be obtaineddirectly from hybridomas which express the antibody, or can be clonedand recombinantly expressed in a host cell (e.g., a CHO cell, or alymphocytic cell). Further examples of host cells are microorganisms,such as E. coli, and fungi, such as yeast. Alternatively, they can beproduced recombinantly in a transgenic non-human animal or plant.However, the present invention also envisions embodiments wherein theantibodies are produced by immunization or vaccination usingimmunization strategies as disclosed herein in situ in a patient.

The present invention also relates to nucleic acids comprising genes ornucleic acid sequences encoding antibodies or parts thereof, e.g. anantibody chain, as described herein. The nucleic acids may be comprisedin a vector, e.g., a plasmid, cosmid, virus, bacteriophage or anothervector used e.g. conventionally in genetic engineering. The vector maycomprise further genes such as marker genes which allow for theselection of the vector in a suitable host cell and under suitableconditions. Furthermore, the vector may comprise expression controlelements allowing proper expression of the coding regions in suitablehosts. Such control elements are known to the artisan and may include apromoter, a splice cassette, and a translation initiation codon.

Preferably, the nucleic acid of the invention is operatively attached tothe above expression control sequences allowing expression in eukaryoticor prokaryotic cells. Control elements ensuring expression in eukaryoticor prokaryotic cells are well known to those skilled in the art.

Methods for construction of nucleic acid molecules according to thepresent invention, for construction of vectors comprising the abovenucleic acid molecules, for introduction of the vectors intoappropriately chosen host cells, for causing or achieving the expressionare well-known in the art.

A further aspect of the present invention relates to a host cellcomprising a nucleic acid or vector as disclosed herein.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence alignment of CLDN3, CLDN4, CLDN6 and CLDN9.

FIG. 2. Immunofluorescence analysis of sera obtained from mice immunizedto produce CLDN6-specific antibodies.

(A) Unfixed CHO-K1 cells co-transfected with nucleic acids encodinghuman CLDN6 and GFP, respectively, were probed with an anti-CLDN6monoclonal mouse antibody (R&D Systems, MAB3656). CLDN6 is located atthe plasma membrane of transfected cells and can be targeted on livingcells by specific antibodies.

(B) Serum from a mouse on the basis of which the hybridoma F3-6C3-H8 wasproduced contained antibodies binding to CLDN6 on the surface of unfixedCHO-K1 cells co-transfected with nucleic acids encoding human CLDN6 andGFP.

FIG. 3. Western blot analysis for assaying endogenous expression ofclaudin proteins in HEK293T cells.

Protein lysates of HEK293T cells transfected with nucleic acids encodingCLDN3, CLDN4, CLDN6, and CLDN9, respectively, or mock-transfected weretested by Western blotting using commercially available anti-CLDN3(A)(Invitrogen, Cat No. 34-1700), anti-CLDN4(A) (Zymed, 32-9400),anti-CLDN6(A) (ARP, 01-8865) and anti-CLDN9(A) (Santa Cruz, sc-17672)antibodies. The antibodies detected expression of their correspondingtargets only in the respective HEK293T transfectants. No endogenousexpression of any of these claudins was observed in non-transfectedHEK293T cells.

FIG. 4. Flow cytometry analysis for assaying the specificity ofcommercially available anti-CLDN antibodies.

Binding of commercially available anti-CLDN antibodies to HEK293T cellstransfected with nucleic acids encoding CLDN3, CLDN4, CLDN6, and CLDN9,respectively, or non-transfected was determined by flow cytometry. Onlythe commercially available anti-CLDN3 antibody is specific for itstarget.

FIG. 5. Flow cytometry analysis for assaying the specificity ofanti-CLDN antibodies prepared according to the invention.

Binding of antibodies in supernatants from monoclonal hybridomasubclones to HEK293T cells co-transfected with a vector encoding CLDN6,CLDN3, CLDN4 or CLDN9 and a vector encoding a fluorescence marker wasdetermined by flow cytometry.

(A) Antibodies in the supernatant from the monoclonal hybridoma subcloneF3-6C3-H8 specifically bind to CLDN6 transfected cells but not to cellstransfected with CLDN3, CLDN4 and CLDN9, respectively. In contrast,antibodies in the supernatant from the monoclonal hybridoma subcloneF4-4F7-F2 bind to cells transfected with CLDN6 or CLDN9. Antibodies inthe supernatant from the monoclonal hybridoma subclone F3-6C3-H8 alsobind to cells transfected with the (1143V)-SNP variant of CLDN6.

(B) Antibodies in the supernatant from the monoclonal hybridoma subcloneF3-7B3-B4 bind to cells transfected with CLDN6, CLDN3 or CLDN9.Antibodies in the supernatant from the monoclonal hybridoma subcloneF3-3F7-A5 bind to cells transfected with CLDN6, CLDN4 or CLDN9.

FIG. 6. Binding specificity of anti-CLDN6 murine monoclonlal antibodiesmuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A.

The binding of anti-CLDN6 antibodies to human CLDN6, 3, 4, 9 and theCLDN6 SNP (single nucleotide polymorphism) variant I143V was analyzed byflow cytometry using HEK293T cells transiently expressing thecorresponding human claudin. HEK293T were co-transfected with afluorescence marker to distinguish between non-transfected (Q1 and Q3population) and transfected (Q2 and Q4 population) cells. The antibodyconcentration used was the concentration that saturated binding to CLDN6(25 μg/ml). The expression of human CLDN6, 3, 4, 9 and CLDN6-SNP(1143V)was confirmed with commercially available monoclonal antibodies againsthuman Claudin-6 (R&D Systems, MAB3656), human Claudin-3 (R&D Systems,MAB4620) and human Claudin-4 (R&D Systems, MAB 4219).

FIG. 7. Relative affinities of anti-CLDN6 murine monoclonal antibodiesmuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A.

To determine relative affinities the binding of anti-CLDN6 antibodies tohuman CLDN6 stably expressed on the surface of HEK293 cells was analyzedby flow cytometry. In the saturation binding experiment theconcentration of the antibodies was plotted against the FACS signals(median of fluorescence intensity). The EC50 (antibody concentrationthat binds to half the binding sites at equilibrium) was calculated bynonlinear regression. The CLDN6-specific antibodies muMAB 59A, 60A, 61D,64A, 65A, 66B and 67A exhibited very low EC50 values (EC50 200-500ng/ml) and saturation of binding was achieved at low concentrations.

FIG. 8. Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6murine monoclonal antibody muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A.

The CDC activity of anti-CLDN6 antibodies was analyzed using aluciferase-dependent assay to detect endogenous ATP within non-lysedcells. Therefore, CHO-K1 cells stably expressing human CLDN6 weretreated with different concentrations of muMAB 59A, 60A, 61D, 64A, 65A,66B and 67A. MuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A exhibiteddose-dependent CDC activity and induced CDC at low concentrations.

FIG. 9. Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6murine monoclonal antibodies muMAB 65A and 66B on endogenously CLDN6expressing NEC8 and NEC8 LVTS2 54 (CLDN6 knock-down) cells.

The anti-CLDN6 antibodies muMAB 65A and 66B induced CDC on NEC8 cells ina dose dependent manner. Target specificity of muMAB 65A and 66B wasproved by using NEC8 LVTS2 54 cells (CLDN6 knock-down).

FIG. 10. Therapeutic effect of muMAB 59A, 60A, 61D, 64A, 65A, 66B and67A in an early treatment xenograft model using mice engrafted with thetumor cell line NEC8.

The model used endogenously CLDN6 expressing NEC8 xenografts in athymicNude-Foxn1^(nu) mice. Compared to the saline control group muMAB 59A,60A, 61D, 64A, 65A, 66B and 67A showed tumor growth inhibition in miceengrafted with NEC8 cells.

FIG. 11. Binding specificity of anti-CLDN6 chimeric monoclonalantibodies chimAB 61D, 64A, 67A and 89A.

The binding of anti-CLDN6 antibodies to human CLDN6, 3, 4 and 9,respectively, was analyzed by flow cytometry using HEK293 cells stablyexpressing the corresponding human claudin. The antibody concentrationused was the concentration that saturated binding (25 μg/ml). Theexpression of human CLDN3, 4, 6 and 9 was confirmed with commerciallyavailable monoclonal antibodies against human Claudin-3 (R&D Systems,MAB4620) and human Claudin-4 (R&D Systems, MAB 4219), and theCLDN6/9-reactive murine monoclonal antibody muMAB 5F2D2, respectively.The negative control was carried out under identical conditions withoutprimary antibody.

FIG. 12. Relative affinities of anti-CLDN6 chimeric monoclonalantibodies chimAB 61D, 64A, 67A and 89A to HEK293-CLDN6 cells.

To determine relative affinities the binding of anti-CLDN6 antibodies tohuman CLDN6 stably expressed on the surface of HEK293 cells was analyzedby flow cytometry. In the saturation binding experiment theconcentration of the antibodies was plotted against the FACS signals(median of fluorescence intensity). The EC50 (antibody concentrationthat binds to half the binding sites at equilibrium) was calculated bynonlinear regression. The CLDN6-specific antibodies chimAB 64A and 89Aexhibited very low EC50 values (EC50 450-600 ng/ml) and saturation ofbinding was achieved at low concentrations. ChimAB 67A and 61D showedlow (EC50 1000 ng/ml) and medium (EC50 2300 ng/ml) EC50 values,respectively.

FIG. 13. Relative affinities of anti-CLDN6 chimeric monoclonalantibodies chimAB 61D, 64A, 67A and 89A to NEC8 cells.

To determine the binding affinities of anti-CLDN6 antibodies to tumorcells that endogenously express human CLDN6 binding to the testicularcancer cell line NEC8 was analyzed by flow cytometry. The CLDN6-specificantibodies chimAB 64A and 89A exhibited very low EC50 values (EC50600-650 ng/ml) and saturation of binding was achieved at lowconcentrations, whereas chimAB 61D and 67A showed medium (EC50 1700ng/ml) and high (EC50 6100 ng/ml) EC50 values, respectively.

FIG. 14. Relative affinities of anti-CLDN6 chimeric monoclonalantibodies chimAB 61D, 64A, 67A and 89A to OV90 cells.

To determine the binding affinities of anti-CLDN6 antibodies to tumorcells that endogenously express human CLDN6 binding to the ovariancancer cell line OV90 was analyzed by flow cytometry. The CLDN6-specificantibodies chimAB 64A and 89A exhibited very low EC50 values (EC50550-600 ng/ml) and saturation of binding was achieved at lowconcentrations. ChimAB 61D and 67A showed medium EC50 values (EC50 1500ng/ml and EC50 2300 ng/ml, respectively).

FIG. 15. Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89A on NEC8wildtype and NEC8 knock-down cells.

The CDC activity of anti-CLDN6 antibodies was analyzed using aluciferase-dependent assay to detect endogenous ATP within non-lysedcells. Therefore, NEC8 wildtype cells (NEC8 LVTS2 77) ectopicallyexpressing luciferase were treated with different concentrations ofchimAB 61D, 64A, 67A and 89A. On NEC-8 cells chimAB 61D, 64A, 67A and89A exhibited CDC activity in a dose-dependent manner, whereas on NEC-8CLDN6 knock-down cells (NEC8 LVTS2 54) none of these antibodies inducedunspecific cell lysis. This result demonstrated target specific effectorfunctions of chimAB 61D, 64A, 67A and 89A.

FIG. 16. Antibody-dependent cellular cytotoxicity (ADCC) activity ofanti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89Aon NEC8 wildtype and NEC8 knock-down cells.

The ADCC activity of anti-CLDN6 antibodies was analyzed using aluciferase-dependent assay to detect endogenous ATP within non-lysedcells. Therefore, NEC-8 wildtype cells (NEC8 LVTS2 77) were treated withdifferent concentrations of chimAB 61D, 64A, 67A and 89A. ChimAB 61D,64A, 67A and 89A exhibited dose-dependent ADCC activity and induced ADCCeven at low antibody concentrations. To demonstrate target specificityNEC8 cells with a stable CLDN6 knock-down (NEC8 LVTS2 54) were used.

FIG. 17. Therapeutic long term effect of anti-CLDN6 murine monoclonalantibodies muMAB 61D, 64A and 67A in an early treatment xenograft modelusing mice engrafted with the tumor cell line NEC8.

The model used endogenously CLDN6 expressing NEC8 xenografts in athymicNude-Foxn1^(nu) mice. Mice were treated for 46 days with CLDN6 specificantibodies. After treatment, the tumor growth was monitored for 60 days.Even after stopping the immunotherapy mice treated with murinemonoclonal antibodies muMAB 61D, 64A and 67A did not show any tumorgrowth.

FIG. 18. Therapeutic effect of the anti-CLDN6 murine monoclonal antibodymuMAB 89A in an early treatment xenograft model using mice engraftedwith the tumor cell line NEC8.

The model used endogenously CLDN6 expressing NEC8 xenografts in athymicNude-Foxn1^(nu) mice. Scatter blots represent volumes of engraftedtumors at different time points during early treatment of NEC8xenografts in athymic Nude-Foxn1^(nu) mice. Compared to the salinecontrol group muMAB 89A showed tumor growth inhibition in mice engraftedwith NEC8 cells (A). Mice were treated for 47 days with PBS as a controland the CLDN6 specific antibody, respectively. The tumor growth wasmonitored for additional 51 days. Compared to the PBS control there wereno tumors detectable in mice treated with muMAB89A at the end of thestudy (B).

FIG. 19. Therapeutic effect of the anti-CLDN6 murine monoclonal antibodymuMAB 64A in an advanced treatment xenograft model using mice engraftedwith the tumor cell line NEC8.

Scatter blots represent volumes of engrafted tumors at different timepoints during treatment of advanced NEC8 xenografts in athymicNude-Foxn1^(nu) mice. Immunotherapy with the murine monoclonalanti-CLDN6 antibody muMAB 64A showed an inhibition of tumor growth ofsolid NEC8 xenografts compared to both the antibody and saline controlgroups.

FIG. 20. Therapeutic long term effect of the anti-CLDN6 murinemonoclonal antibody muMAB 64A in an advanced treatment xenograft modelusing mice engrafted with the tumor cell line NEC8.

15 days after engraftment mice were treated for 45 days with the CLDN6specific antibody muMAB 64A. The tumor growth was monitored foradditional 49 days (A). The survival plot showed prolonged survival ofmice treated with the CLDN6 specific antibody muMAB 64A (B).

FIG. 21. Therapeutic effect of anti-CLDN6 murine monoclonal antibodiesmuMAB 61D, 67A and 89A in an advanced treatment xenograft model usingmice engrafted with the tumor cell line NEC8.

Scatter blots represent volumes of engrafted NEC8 tumors at differenttime points during treatment of advanced NEC8 xenografts. Compared tothe saline and antibody control groups the inhibition of tumor growthwas achieved with the murine monoclonal anti-CLDN6 antibodies muMAB 61D,67A and 89A.

FIG. 22. Therapeutic long term effect of anti-CLDN6 murine monoclonalantibodies muMAB 61D, 67A and 89A in an advanced treatment xenograftmodel using mice engrafted with the tumor cell line NEC8.

17 days after engraftment mice were treated for 42 days with the CLDN6specific antibodies muMAB 61D, 67A and 89A. The tumor growth has beenmonitored for additional 49 days (A). The survival plot showed prolongedsurvival of mice treated with the CLDN6 specific antibodies muMAB 61Dand 67A (B).

FIG. 23. Therapeutic effect of anti-CLDN6 murine monoclonal antibodiesmuMAB 64A and 89A in an advanced treatment xenograft model using miceengrafted with NEC8 wildtype and NEC8 cells with a stable CLDN6knock-down.

MuMAB 64A and 89A only show therapeutic effect in mice engrafted withNEC8 wildtype but not in mice engrafted with NEC8 CLDN6 knock-down cellsdemonstrating target-specificity of the antibodies in vivo.

FIG. 24. High resolution epitope-mapping of chimMAB 61D, 64A, 67A and89A.

Alanine mutants are named as ‘wildtype residue number alanine’ or‘wildtype residue number glycine’ in case of wildtype-alanine, where theamino acids are given in the single-letter code. The amino acids F35,G37, S39 and possibly T33 of the first extracellular domain of CLDN6 areimportant for the interaction with the CLDN6 specific chimericantibodies chimAB 61D, 64A, 67A and 89A. Residue 140 is essential forthe binding of chimAB 89A and it contributes to the binding of chimAB61D and 67A. In addition, L151 of the second extracellular domain ofCLDN6 contributes to the interaction with chimAB 67A. Althoughimmunofluorescence experiments confirmed the expression of CLDN6 mutantsP28A, W30A, G49A, L50A, W51A, C54A and C64A they did not show membranousstaining. For this reason we cannot exclude interaction of ourantibodies with these amino acids. Altogether, the epitope as identifiedhere is consistent with our immunization strategy using DNA and peptidesof the EC1 domain of CLDN6.

FIG. 25. Alignment of heavy chain variable region amino acid sequencesof CLDN6 specific antibodies of the invention.

The CDR sequences (HCDR1, HCDR2, and HCDR3) are outlined by a box.

FIG. 26. Alignment of light chain variable region amino acid sequencesof CLDN6 specific antibodies of the invention.

The CDR sequences (LCDR1, LCDR2, and LCDR3) are outlined by a box.

DEFINITION OF TERMS

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps. The terms “a” and “an” and “the”and similar reference used in the context of describing the invention(especially in the context of the claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. Recitation of ranges of values hereinis merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”),provided herein is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of theinvention.

Claudins are a family of proteins that are the most important componentsof tight junctions, where they establish the paracellular barrier thatcontrols the flow of molecules in the intercellular space between cellsof an epithelium. Claudins are transmembrane proteins spanning themembrane 4 times with the N-terminal and the C-terminal end both locatedin the cytoplasm. The first extracellular loop consists on average of 53amino acids and the second one of around 24 amino acids. CLDN6 and CLDN9are the most similar members of the CLDN family.

The term “CLDN” as used herein means claudin and includes CLDN6, CLDN9,CLDN4 and CLDN3. Preferably, a CLDN is a human CLDN.

The term “CLDN6” preferably relates to human CLDN6, and, in particular,to (i) a nucleic acid comprising a nucleic acid sequence encoding theamino sequence of SEQ ID NO: 2 or encoding the amino sequence of SEQ IDNO: 8 such as a nucleic acid comprising the nucleic acid sequence of SEQID NO: 1 or (ii) a protein comprising the amino acid sequence of SEQ IDNO: 2 or comprising the amino acid sequence of SEQ ID NO: 8. The firstextracellular loop of CLDN6 preferably comprises amino acids 28 to 80,more preferably amino acids 28 to 76 of the amino acid sequence shown inSEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 8, such asthe amino acid sequence shown in SEQ ID NO: 7. The second extracellularloop of CLDN6 preferably comprises amino acids 138 to 160, preferablyamino acids 141 to 159, more preferably amino acids 145 to 157 of theamino acid sequence shown in SEQ ID NO: 2 or the amino acid sequenceshown in SEQ ID NO: 8, such as the amino acid sequence shown in SEQ IDNO: 6. Said first and second extracellular loops preferably form theextracellular portion of CLDN6.

The term “CLDN9” preferably relates to human CLDN9, and, in particular,to (i) a nucleic acid comprising a nucleic acid sequence encoding theamino sequence of SEQ ID NO: 9 or (ii) a protein comprising the aminoacid sequence of SEQ ID NO: 9. The first extracellular loop of CLDN9preferably comprises amino acids 28 to 76 of the amino acid sequenceshown in SEQ ID NO: 9. The second extracellular loop of CLDN9 preferablycomprises amino acids 141 to 159 of the amino acid sequence shown in SEQID NO: 9. Said first and second extracellular loops preferably form theextracellular portion of CLDN9.

The term “CLDN4” preferably relates to human CLDN4, and, in particular,to (i) a nucleic acid comprising a nucleic acid sequence encoding theamino sequence of SEQ ID NO: 10 or (ii) a protein comprising the aminoacid sequence of SEQ ID NO: 10. The first extracellular loop of CLDN4preferably comprises amino acids 28 to 76 of the amino acid sequenceshown in SEQ ID NO: 10. The second extracellular loop of CLDN4preferably comprises amino acids 141 to 159 of the amino acid sequenceshown in SEQ ID NO: 10. Said first and second extracellular loopspreferably form the extracellular portion of CLDN4.

The term “CLDN3” preferably relates to human CLDN3, and, in particular,to (i) a nucleic acid comprising a nucleic acid sequence encoding theamino sequence of SEQ ID NO: 11 or (ii) a protein comprising the aminoacid sequence of SEQ ID NO: 11. The first extracellular loop of CLDN3preferably comprises amino acids 27 to 75 of the amino acid sequenceshown in SEQ ID NO: 11. The second extracellular loop of CLDN3preferably comprises amino acids 140 to 158 of the amino acid sequenceshown in SEQ ID NO: 11. Said first and second extracellular loopspreferably form the extracellular portion of CLDN3.

The above described CLDN sequences include any variants of saidsequences, in particular mutants, splice variants, conformations,isoforms, allelic variants, species variants and species homologs, inparticular those which are naturally present. An allelic variant relatesto an alteration in the normal sequence of a gene, the significance ofwhich is often unclear. Complete gene sequencing often identifiesnumerous allelic variants for a given gene. A species homolog is anucleic acid or amino acid sequence with a different species of originfrom that of a given nucleic acid or amino acid sequence. The term“CLDN” shall encompass (i) CLDN splice variants, (ii)CLDN-posttranslationally modified variants, particularly includingvariants with different glycosylation such as N-glycosylation status,(iii) CLDN conformation variants, (iv) CLDN cancer related and CLDNnon-cancer related variants. Preferably, a CLDN is present in its nativeconformation.

CLDN6 has been found to be expressed, for example, in ovarian cancer,lung cancer, gastric cancer, breast cancer, hepatic cancer, pancreaticcancer, skin cancer, melanomas, head neck cancer, sarcomas, bile ductcancer, renal cell cancer, and urinary bladder cancer. CLDN6 is aparticularly preferred target for the prevention and/or treatment ofovarian cancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer, agerm cell tumor such as a teratocarcinoma or an embryonal carcinoma, inparticular a germ cell tumor of the testis, and the metastatic formsthereof. In one embodiment, the cancer disease associated with CLDN6expression is selected from the group consisting of ovarian cancer, lungcancer, metastatic ovarian cancer and metastatic lung cancer.Preferably, the ovarian cancer is a carcinoma or an adenocarcinoma.Preferably, the lung cancer is a carcinoma or an adenocarcinoma, andpreferably is bronchiolar cancer such as a bronchiolar carcinoma orbronchiolar adenocarcinoma. In one embodiment, the tumor cell associatedwith CLDN6 expression is a cell of such a cancer.

The term “portion” refers to a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence.

The terms “part” and “fragment” are used interchangeably herein andrefer to a continuous element. For example, a part of a structure suchas an amino acid sequence or protein refers to a continuous element ofsaid structure. A portion, a part or a fragment of a structurepreferably comprises one or more functional properties of saidstructure. For example, a portion, a part or a fragment of an epitope orpeptide is preferably immunologically equivalent to the epitope orpeptide it is derived from.

The term “an extracellular portion of a CLDN” in the context of thepresent invention refers to a part of a CLDN facing the extracellularspace of a cell and preferably being accessible from the outside of saidcell, e.g., by antibodies located outside the cell. Preferably, the termrefers to one or more extracellular loops or a part thereof or any otherextracellular part of a CLDN which is preferably specific for said CLDN.Preferably, said part comprises at least 5, at least 8, at least 10, atleast 15, at least 20, at least 30, or at least 50 amino acids or more.

The term “CLDN associated with the surface of a cell” is to beunderstood to relate to native CLDN, i.e. CLDN in its non-denatured,preferably naturally folded state. Preferably, the term “CLDN associatedwith the surface of a cell” means that the CLDN is associated with andlocated at the plasma membrane of said cell, wherein at least a part ofthe CLDN, preferably the extracellular portion, faces the extracellularspace of said cell and is accessible from the outside of said cell,e.g., by antibodies located outside the cell. The association may bedirect or indirect. For example, the association may be by one or moretransmembrane domains, one or more lipid anchors, and/or by theinteraction with any other protein, lipid, saccharide, or otherstructure that can be found on the outer leaflet of the plasma membraneof a cell. For example, a CLDN associated with the surface of a cell maybe a transmembrane protein, i.e. an integral membrane protein, having anextracellular portion or may be a protein associated with the surface ofa cell by interacting with another protein that is a transmembraneprotein.

CLDN6 is associated with the surface of a cell if it is located at thesurface of said cell and is accessible to binding by CLDN6-specificantibodies added to the cell. In preferred embodiments, a cell beingcharacterized by association of CLDN6 with its cell surface is a cellexpressing CLDN6. It is to be understood that in the case where CLDN6 isexpressed by cells, the CLDN6 associated with the surface of said cellsmay only be a portion of the expressed CLDN6.

The term “a cell carrying a CLDN” preferably means that said cellcarries a CLDN on its surface, i.e., that the CLDN is associated withthe surface of said cell.

“Cell surface” or “surface of a cell” is used in accordance with itsnormal meaning in the art, and thus includes the outside of the cellwhich is accessible to binding by proteins and other molecules.

The expression “CLDN expressed on the surface of a cell” means that theCLDN expressed by a cell is found in association with the surface ofsaid cell.

According to the invention CLDN6 is not substantially expressed in acell and is not substantially associated with a cell surface if thelevel of expression and association is lower compared to expression andassociation in placenta cells or placenta tissue. Preferably, the levelof expression and association is less than 10%, preferably less than 5%,3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression and association inplacenta cells or placenta tissue or even lower. Preferably, CLDN6 isnot substantially expressed in a cell and is not substantiallyassociated with a cell surface if the level of expression andassociation exceeds the level of expression and association innon-tumorigenic, non-cancerous tissue other than placenta tissue by nomore than 2-fold, preferably 1.5-fold, and preferably does not exceedthe level of expression and association in said non-tumorigenic,non-cancerous tissue. Preferably, CLDN6 is not substantially expressedin a cell and is not substantially associated with a cell surface if thelevel of expression or association is below the detection limit and/orif the level of expression or association is too low to allow binding byCLDN6-specific antibodies added to the cells.

According to the invention CLDN6 is expressed in a cell and isassociated with a cell surface if the level of expression andassociation exceeds the level of expression and association innon-tumorigenic, non-cancerous tissue other than placenta tissue,preferably by more than 2-fold, preferably 10-fold, 100-fold, 1000-fold,or 10000-fold. Preferably, CLDN6 is expressed in a cell and isassociated with a cell surface if the level of expression andassociation is above the detection limit and/or if the level ofexpression and association is high enough to allow binding byCLDN6-specific antibodies added to the cells. Preferably, CLDN6expressed in a cell is expressed or exposed on the surface of said cell.

The term “raft” refers to the sphingolipid- and cholesterol-richmembrane microdomains located in the outer leaflet area of the plasmamembrane of a cell. The ability of certain proteins to associate withinsuch domains and their ability of forming “aggregates” or “focalaggregates” can effect the protein's function. For example, thetranslocation of CLDN6 molecules into such structures, after being boundby antibodies of the present invention, creates a high density of CLDN6antigen-antibody complexes in the plasma membranes. Such a high densityof CLDN6 antigen-antibody complexes can enable efficient activation ofthe complement system during CDC.

According to the invention, the term “disease” refers to anypathological state, including cancer, in particular those forms ofcancer described herein.

“Diseases involving cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface” means according to theinvention that expression and association in cells of a diseased tissueor organ is preferably increased compared to the state in a healthytissue or organ. An increase refers to an increase by at least 10%, inparticular at least 20%, at least 50%, at least 100%, at least 200%, atleast 500%, at least 1000%, at least 10000% or even more. In oneembodiment, expression and association with the cell surface is onlyfound in a diseased tissue, while expression in a healthy tissue isrepressed. According to the invention, diseases associated with cellsexpressing CLDN6 and being characterized by association of CLDN6 withtheir cell surface include tumor diseases such as cancer diseases.Furthermore, according to the invention, tumor diseases such as cancerdiseases preferably are those wherein the tumor cells or cancer cellsexpress CLDN6 and are characterized by association of CLDN6 with theircell surface.

As used herein, a “tumor disease”, “tumor-related disease” or“tumorigenic disease” includes a disease characterized by aberrantlyregulated cellular growth, proliferation, differentiation, adhesion,and/or migration, which may result in the production of or tendency toproduce tumors and/or tumor metastasis. By “tumor cell” is meant anabnormal cell that grows by a rapid, uncontrolled cellular proliferationand continues to grow after the stimuli that initiated the new growthcease.

By “tumor” is meant an abnormal group of cells or a tissue growing by arapid, uncontrolled cellular proliferation and continues to grow afterthe stimuli that initiated the new growth cease. Tumors show partial orcomplete lack of structural organization and functional coordinationwith the normal tissue, and usually form a distinct mass of tissue,which may be either benign, pre-malignant or malignant.

Preferably, a “tumor disease”, “tumor-related disease” or “tumorigenicdisease” according to the invention is a cancer disease, i.e. amalignant disease and a tumor cell is a cancer cell. Preferably, a“tumor disease”, “tumor-related disease” or “tumorigenic disease” ischaracterized by cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface and a tumor cell expressesCLDN6 and is characterized by association of CLDN6 with its cellsurface.

A cell expressing CLDN6 and being characterized by association of CLDN6with its cell surface preferably is a tumor cell or cancer cell,preferably of the tumors and cancers described herein. Preferably, suchcell is a cell other than a placental cell.

Preferred cancer diseases or cancers according to the invention areselected from the group consisting of ovarian cancer, in particularovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer,including small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma,gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skincancer, in particular basal cell carcinoma and squamous cell carcinoma,malignant melanoma, head and neck cancer, in particular malignantpleomorphic adenoma, sarcoma, in particular synovial sarcoma andcarcinosarcoma, bile duct cancer, cancer of the urinary bladder, inparticular transitional cell carcinoma and papillary carcinoma, kidneycancer, in particular renal cell carcinoma including clear cell renalcell carcinoma and papillary renal cell carcinoma, colon cancer, smallbowel cancer, including cancer of the ileum, in particular small boweladenocarcinoma and adenocarcinoma of the ileum, testicular embryonalcarcinoma, placental choriocarcinoma, cervical cancer, testicularcancer, in particular testicular seminoma, testicular teratoma andembryonic testicular cancer, uterine cancer, a germ cell tumor such as ateratocarcinoma or an embryonal carcinoma, in particular a germ celltumor of the testis, and the metastatic forms thereof.

The main types of lung cancer are small cell lung carcinoma (SCLC) andnon-small cell lung carcinoma (NSCLC). There are three main sub-types ofthe non-small cell lung carcinomas: squamous cell lung carcinoma,adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas accountfor approximately 10% of lung cancers. This cancer usually is seenperipherally in the lungs, as opposed to small cell lung cancer andsquamous cell lung cancer, which both tend to be more centrally located.

Skin cancer is a malignant growth on the skin. The most common skincancers are basal cell cancer, squamous cell cancer, and melanoma.Malignant melanoma is a serious type of skin cancer. It is due touncontrolled growth of pigment cells, called melanocytes.

According to the invention, a “carcinoma” is a cancer that begins in thelining layer (epithelial cells) of organs.

“Bronchiolar carcinoma” is a carcinoma of the lung, thought to bederived from epithelium of terminal bronchioles, in which the neoplastictissue extends along the alveolar walls and grows in small masses withinthe alveoli. Mucin may be demonstrated in some of the cells and in thematerial in the alveoli, which also includes denuded cells.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so. Ovarianadenocarcinoma is the most common type of ovarian carcinoma. It includesthe serous and mucinous adenocarcinomas, the clear cell adenocarcinomaand the endometrioid adenocarcinoma.

“Cystadenocarcinoma” is a malignant form of a surface epithelial-stromaltumor, a type of ovarian cancer.

Surface epithelial-stromal tumors are a class of ovarian neoplasms thatare thought to be derived from the ovarian surface epithelium (modifiedperitoneum) or from ectopic endometrial or Fallopian tube (tubal)tissue. This group of tumors accounts for the majority of all ovariantumors.

Teratocarcinoma refers to a germ cell tumor that is a mixture ofteratoma with embryonal carcinoma, or with choriocarcinoma, or withboth. Choriocarcinoma is a malignant, trophoblastic and aggressivecancer, usually of the placenta. It is characterized by earlyhematogenous spread to the lungs.

A sarcoma is a cancer of the connective tissue (bone, cartilage, fat)resulting in mesoderm proliferation. This is in contrast to carcinomas,which are of epithelial origin. A synovial sarcoma is a rare form ofcancer which usually occurs near to the joints of the arm or leg. It isone of the soft tissue sarcomas.

Renal cell carcinoma also known as renal cell cancer or renal celladenocarcinoma is a kidney cancer that originates in the lining of theproximal convoluted tubule, the very small tubes in the kidney thatfilter the blood and remove waste products. Renal cell carcinoma is byfar the most common type of kidney cancer in adults and the most lethalof all the genitourinary tumors. Distinct subtypes of renal cellcarcinoma are clear cell renal cell carcinoma and papillary renal cellcarcinoma. Clear cell renal cell carcinoma is the most common form ofrenal cell carcinoma. When seen under a microscope, the cells that makeup clear cell renal cell carcinoma appear very pale or clear. Papillaryrenal cell carcinoma is the second most common subtype. These cancersform little finger-like projections (called papillae) in some, if notmost, of the tumors.

A germ cell tumor is a neoplasm derived from germ cells. Germ celltumors can be cancerous or non-cancerous tumors. Germ cells normallyoccur inside the gonads (ovary and testis). Germ cell tumors thatoriginate outside the gonads (e.g. in head, inside the mouth, neck,pelvis; in fetuses, babies, and young children most often found on thebody midline, particularly at the tip of the tailbone) may be birthdefects resulting from errors during development of the embryo.

The two major classes of germ cell tumors are the seminomas andnon-seminomas, wherein non-seminomas include: teratocarcinoma, embryonalcarcinoma, yolk sac tumors, choriocarcinoma and differentiated teratoma.Most cell lines from non-seminomas are equivalent to embryonalcarcinomas, that is, they are composed almost entirely of stem cellswhich do not differentiate under basal conditions, though some mayrespond to inducers of differentiation such as retinoic acid.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if ovarian cancermetastasizes to the liver, the secondary tumor is made up of abnormalovarian cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic ovarian cancer, not liver cancer.

By “treat” is meant to administer a compound or composition as describedherein to a subject in order to prevent or eliminate a disease,including reducing the size of a tumor or the number of tumors in asubject; arrest or slow a disease in a subject; inhibit or slow thedevelopment of a new disease in a subject; decrease the frequency orseverity of symptoms and/or recurrences in a subject who currently hasor who previously has had a disease; and/or prolong, i.e. increase thelifespan of the subject.

The term “treatment of a disease” includes curing, shortening theduration, ameliorating, preventing, slowing down or inhibitingprogression or worsening, or preventing or delaying the onset of adisease or the symptoms thereof.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing adisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a disease, inparticular cancer is a subject who has an increased risk for developinga disease, as such a subject may continue to develop a disease. Subjectswho currently have, or who have had, a cancer also have an increasedrisk for cancer metastases.

The term “immunotherapy” relates to a treatment involving a specificimmune reaction. In the context of the present invention, terms such as“protect”, “prevent”, “prophylactic”, “preventive”, or “protective”relate to the prevention or treatment or both of the occurrence and/orthe propagation of a tumor in an individual. The term “immunotherapy” inthe context of the present invention preferably refers to active tumorimmunization or tumor vaccination. A prophylactic administration of animmunotherapy, for example, a prophylactic administration of thecomposition of the invention, preferably protects the recipient from thedevelopment of tumor growth. A therapeutic administration of animmunotherapy, for example, a therapeutic administration of thecomposition of the invention, may lead to the inhibition of theprogress/growth of the tumor. This comprises the deceleration of theprogress/growth of the tumor, in particular a disruption of theprogression of the tumor, which preferably leads to elimination of thetumor. A therapeutic administration of an immunotherapy may protect theindividual, for example, from the dissemination or metastasis ofexisting tumors.

The term “immunization” or “vaccination” describes the process ofadministering antigen to a subject with the purpose of inducing animmune response for therapeutic or prophylactic reasons.

The terms “subject”, “individual”, “organism” or “patient” are usedinterchangeably and relate to vertebrates, preferably mammals. Forexample, mammals in the context of the present invention are humans,non-human primates, domesticated animals such as dogs, cats, sheep,cattle, goats, pigs, horses etc., laboratory animals such as mice, rats,rabbits, guinea pigs, etc. as well as animals in captivity such asanimals of zoos. The term “animal” as used herein also includes humans.The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease, preferably a disease associatedwith expression of CLDN6, preferably a tumorigenic disease such as acancer.

The term “adjuvant” relates to compounds which prolongs or enhances oraccelerates an immune response. The composition of the present inventionpreferably exerts its effect without addition of adjuvants. Still, thecomposition of the present application may contain any known adjuvant.Adjuvants comprise a heterogeneous group of compounds such as oilemulsions (e.g., Freund's adjuvants), mineral compounds (such as alum),bacterial products (such as Bordetella pertussis toxin), liposomes, andimmune-stimulating complexes. Examples for adjuvants aremonophosphoryl-lipid-A (MPL SmithKline Beecham). Saponins such as QS21(SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7,QS17, QS18, and QS-L1 (So et al., 1997, Mol. Cells. 7: 178-186),incomplete Freund's adjuvants, complete Freund's adjuvants, vitamin E,montanid, alum, CpG oligonucleotides (Krieg et al., 1995, Nature 374:546-549), and various water-in-oil emulsions which are prepared frombiologically degradable oils such as squalene and/or tocopherol.

According to the invention, a sample may be any sample useful accordingto the present invention, in particular a biological sample such atissue sample, including bodily fluids, and/or a cellular sample and maybe obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“biological sample” also includes fractions of biological samples.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, and includes any molecule comprising an antigen binding portionthereof. The term “antibody” includes monoclonal antibodies andfragments or derivatives thereof, including, without limitation, humanmonoclonal antibodies, humanized monoclonal antibodies, chimericmonoclonal antibodies, single chain antibodies, e.g., scFv's andantigen-binding antibody fragments such as Fab and Fab′ fragments andalso includes all recombinant forms of antibodies, e.g., antibodiesexpressed in prokaryotes, unglycosylated antibodies, and anyantigen-binding antibody fragments and derivatives as described herein.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. Each lightchain is comprised of a light chain variable region (abbreviated hereinas VL) and a light chain constant region. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system.

According to the invention, the term “at least one of the CDR sequences”preferably means at least the CDR3 sequence. The term “CDR sequences ofan antibody chain” preferably relates to CDR1, CDR2 and CDR3 of theheavy chain or light chain of an antibody.

According to the invention, a reference to an antibody chain comprisinga particular CDR sequence such as a particular CDR3 sequence means thatsaid particular CDR sequence either forms the CDR region such as theCDR3 region of said antibody chain, i.e. the CDR region consists of saidparticular CDR sequence, or forms a part of the CDR region such as theCDR3 region of said antibody chain, i.e. the CDR region comprises saidparticular CDR sequence.

If according to the invention reference is made to an antibodycomprising a particular antibody heavy chain and/or a particularantibody light chain, such as a chain comprising particular CDRsequences, it is preferred that both heavy chains and/or both lightchains of the antibody are each composed of the particular antibodyheavy chain and/or the particular antibody light chain.

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals, while the constant portions are homologous to sequences ofantibodies derived from another. One clear advantage to such chimericforms is that the variable region can conveniently be derived frompresently known sources using readily available B-cells or hybridomasfrom non-human host organisms in combination with constant regionsderived from, for example, human cell preparations. While the variableregion has the advantage of ease of preparation and the specificity isnot affected by the source, the constant region being human, is lesslikely to elicit an immune response from a human subject when theantibodies are injected than would the constant region from a non humansource. However the definition is not limited to this particularexample.

The term “antigen-binding portion” of an antibody (or simply “bindingportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. A further example is binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region. The binding-domainimmunoglobulin fusion proteins are further disclosed in US 2003/0118592and US 2003/0133939. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. In the context of the presentinvention, the epitope is preferably derived from a CLDN protein.Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. An epitope of aprotein such as a CLDN preferably comprises a continuous ordiscontinuous portion of said protein and is preferably between 5 and100, preferably between 5 and 50, more preferably between 8 and 30, mostpreferably between 10 and 25 amino acids in length, for example, theepitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 amino acids in length.

The term “discontinuous epitope” as used herein, means a conformationalepitope on a protein antigen which is formed from at least two separateregions in the primary sequence of the protein.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with (a) a cell surface antigen, and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to CLDN6, and to other targets, such as Fc receptors oneffector cells. The term “bispecific antibodies” also includesdiabodies. Diabodies are bivalent, bispecific antibodies in which the VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

As used herein, the term “heteroantibodies” refers to two or moreantibodies, derivatives thereof, or antigen binding regions linkedtogether, at least two of which have different specificities. Thesedifferent specificities include a binding specificity for an Fc receptoron an effector cell, and a binding specificity for an antigen or epitopeon a target cell, e.g., a tumor cell.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. In one embodiment, the monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from anon-human animal, e.g., mouse, fused to an immortalized cell.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The invention includes all antibodies and derivatives of antibodies asdescribed herein which for the purposes of the invention are encompassedby the term “antibody”. The term “antibody derivatives” refers to anymodified form of an antibody, e.g., a conjugate of the antibody andanother agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLDN6 is substantially free of antibodies that specifically bindantigens other than CLDN6). An isolated antibody that specifically bindsto an epitope, isoform or variant of human CLDN6 may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., CLDN6 species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies relates to antibodies having different specificities andbeing combined in a well defined composition.

According to the present invention, an antibody is capable of binding toa predetermined target if it has a significant affinity for saidpredetermined target and binds to said predetermined target in standardassays such as the assays described herein. Preferably, an antibody iscapable of binding to a target if it detectably binds to said target ina flow cytometry analysis (FACS analysis) wherein binding of saidantibody to said target expressed on the surface of intact cells isdetermined. Preferably, the antibody detectably binds to said target ifpresent in a concentration of 10 μg/ml or lower, 5 μg/ml or lower or 2μg/ml or lower. Preferably, the antibody detectably binds to said targetif present in a concentration of 50 nM or lower, 30 nM or lower or 15 nMor lower. “Affinity” or “binding affinity” is often measured byequilibrium dissociation constant (K_(D)). Preferably, the term“significant affinity” refers to the binding to a predetermined targetwith a dissociation constant (K_(D)) of 10⁻⁵ M or lower, 10⁻⁶ M orlower, 10⁻⁷ M or lower, 10⁻⁸ M or lower, 10⁻⁹M or lower, 10⁻¹⁰ M orlower, 10⁻¹¹ M or lower, or 10⁻¹² M or lower. Antibodies of the presentinvention preferably have EC50 values for binding to CLDN6 of 6500 ng/mlor lower, 3000 ng/ml or lower, 2500 ng/ml or lower, 2000 ng/ml or lower,1500 ng/ml or lower, 1000 ng/ml or lower, 500 ng/ml or lower, 400 ng/mlor lower, 300 ng/ml or lower, 200 ng/ml or lower, or 100 ng/ml or lower.

An antibody is not (substantially) capable of binding to a target if ithas no significant affinity for said target and does not bindsignificantly to said target in standard assays. Preferably, an antibodyis not (substantially) capable of binding to a target if it does notdetectably bind to said target in a flow cytometry analysis (FACSanalysis) wherein binding of said antibody to said target expressed onthe surface of intact cells is determined. Preferably, the antibody doesnot detectably bind to said target if present in a concentration of upto 2 μg/ml, preferably up to 5 μg/ml, preferably up to 10 μg/ml,preferably up to 20 μg/ml, more preferably up to 50 μg/ml, in particularup to 100 μg/ml, or up to 150 μg/ml, up to 200 μg/ml or higher.Preferably, the antibody does not detectably bind to said target ifpresent in a concentration of up to 15 nM, preferably up to 30 nM,preferably up to 50 nM, preferably up to 100 nM, preferably up to 150nM, or up to 170 nM, up to 300 mM, up to 600 nM, up to 1000 nM, up to1300 nM or higher. Preferably, the antibody does not detectably bind tosaid target if present in a concentration that saturates binding to thetarget to which the antibody binds, i.e. CLDN6. Preferably, an antibodyhas no significant affinity for a target if it binds to said target witha K_(D) that is at least 10-fold, 100-fold, 10³-fold, 10⁴-fold,10⁵-fold, or 10⁶-fold higher than the K_(D) for binding to thepredetermined target to which the antibody is capable of binding. Forexample, if the K_(D) for binding of an antibody to the target to whichthe antibody is capable of binding is 10⁻⁷ M, the K_(D) for binding to atarget for which the antibody has no significant affinity would be is atleast 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

An antibody is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not capable of bindingto other targets, i.e. has no significant affinity for other targets anddoes not significantly bind to other targets in standard assays.According to the invention, an antibody is specific for CLDN6 if it iscapable of binding to CLDN6 but is not capable of binding to othertargets, in particular claudin proteins other than CLDN6 such as CLDN9,CLDN4, CLDN3 and CLDN1. Preferably, an antibody is specific for CLDN6 ifthe affinity for and the binding to a claudin protein other than CLDN6such as CLDN9, CLDN4, CLDN3 and CLDN1 does not significantly exceed theaffinity for or binding to claudin-unrelated proteins such as bovineserum albumin (BSA), casein, human serum albumin (HSA) or non-claudintransmembrane proteins such as MHC molecules or transferrin receptor orany other specified polypeptide. Preferably, an antibody is specific fora predetermined target if it binds to said target with a K_(D) that isat least 10-fold, 100-fold, 10³-fold, 10⁴-fold, 10⁵-fold, or 10⁶-foldlower than the K_(D) for binding to a target for which it is notspecific. For example, if the K_(D) for binding of an antibody to thetarget for which it is specific is 10⁻⁷ M, the K_(D) for binding to atarget for which it is not specific would be at least 10⁻⁶ M, 10⁻⁵ M,10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

Binding of an antibody to a target can be determined experimentallyusing any suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W.H.Freeman and Company New York, N.Y. (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

A unique feature of the antibody of the present invention is the abilityto bind cell surface claudin 6. This is demonstrated by flow cytometryanalysis of cells expressing claudin 6.

To test the binding of monoclonal antibodies to live cells expressingclaudins, flow cytometry can be used. Briefly, cell lines expressingmembrane-associated claudins (grown under standard growth conditions)are mixed with various concentrations of antibodies in PBS containing 2%heat inactivated FCS and 0.1% NaN₃ at 4° C. for 30 min. After washing,the cells are reacted with a fluorescently labeled secondary antibodyunder the same conditions as the primary antibody staining. The samplescan be analyzed by FACS using light and side scatter properties to gateon single cells and binding of the labeled antibodies is determined.

The term “binding” according to the invention preferably relates to aspecific binding as defined herein.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA. A nucleic acid molecule can be employed for introduction into, i.e.transfection of, cells, for example, in the form of RNA which can beprepared by in vitro transcription from a DNA template. The RNA canmoreover be modified before application by stabilizing sequences,capping, and polyadenylation.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

Nucleic acids may, according to the invention, be present alone or incombination with other nucleic acids, which may be homologous orheterologous. In preferred embodiments, a nucleic acid is functionallylinked to expression control sequences which may be homologous orheterologous with respect to said nucleic acid wherein the term“homologous” means that the nucleic acid is also functionally linked tothe expression control sequence naturally and the term “heterologous”means that the nucleic acid is not functionally linked to the expressioncontrol sequence naturally.

A nucleic acid, such as a nucleic acid expressing RNA and/or protein orpeptide, and an expression control sequence are “functionally” linked toone another, if they are covalently linked to one another in such a waythat expression or transcription of said nucleic acid is under thecontrol or under the influence of said expression control sequence. Ifthe nucleic acid is to be translated into a functional protein, then,with an expression control sequence functionally linked to a codingsequence, induction of said expression control sequence results intranscription of said nucleic acid, without causing a frame shift in thecoding sequence or said coding sequence not being capable of beingtranslated into the desired protein or peptide.

The term “expression control sequence” comprises according to theinvention promoters, ribosome binding sites, enhancers and other controlelements which regulate transcription of a gene or translation of amRNA. In particular embodiments of the invention, the expression controlsequences can be regulated. The exact structure of expression controlsequences may vary as a function of the species or cell type, butgenerally comprises 5′-untranscribed and 5′- and 3′-untranslatedsequences which are involved in initiation of transcription andtranslation, respectively, such as TATA box, capping sequence, CAATsequence, and the like. More specifically, 5′-untranscribed expressioncontrol sequences comprise a promoter region which includes a promotersequence for transcriptional control of the functionally linked nucleicacid. Expression control sequences may also comprise enhancer sequencesor upstream activator sequences.

According to the invention the term “promoter” or “promoter region”relates to a nucleic acid sequence which is located upstream (5′) to thenucleic acid sequence being expressed and controls expression of thesequence by providing a recognition and binding site for RNA-polymerase.The “promoter region” may include further recognition and binding sitesfor further factors which are involved in the regulation oftranscription of a gene. A promoter may control the transcription of aprokaryotic or eukaryotic gene. Furthermore, a promoter may be“inducible” and may initiate transcription in response to an inducingagent or may be “constitutive” if transcription is not controlled by aninducing agent. A gene which is under the control of an induciblepromoter is not expressed or only expressed to a small extent if aninducing agent is absent. In the presence of the inducing agent the geneis switched on or the level of transcription is increased. This ismediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention includepromoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMVpromoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.According to the invention, the term expression also includes an“aberrant expression” or “abnormal expression”.

“Aberrant expression” or “abnormal expression” means according to theinvention that expression is altered, preferably increased, compared toa reference; preferably compared to the state in a non-tumorigenicnormal cell or a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50% orat least 100%. In one embodiment, expression is only found in a diseasedtissue, while expression in a healthy tissue is repressed.

In a preferred embodiment, a nucleic acid molecule is according to theinvention present in a vector, where appropriate with a promoter, whichcontrols expression of the nucleic acid. The term “vector” is used herein its most general meaning and comprises any intermediary vehicle for anucleic acid which enables said nucleic acid, for example, to beintroduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. Vectors compriseplasmids, phagemids, bacteriophages or viral genomes. The term “plasmid”as used herein generally relates to a construct of extrachromosomalgenetic material, usually a circular DNA duplex, which can replicateindependently of chromosomal DNA.

As the vector for expression of an antibody, either of a vector type inwhich the antibody heavy chain and light chain are present in differentvectors or a vector type in which the heavy chain and light chain arepresent in the same vector can be used.

The teaching given herein with respect to specific nucleic acid andamino acid sequences, e.g. those shown in the sequence listing, is to beconstrued so as to also relate to modifications, i.e. variants, of saidspecific sequences resulting in sequences which are functionallyequivalent to said specific sequences, e.g. amino acid sequencesexhibiting properties identical or similar to those of the specificamino acid sequences and nucleic acid sequences encoding amino acidsequences exhibiting properties identical or similar to those of theamino acid sequences encoded by the specific nucleic acid sequences. Oneimportant property is to retain binding of an antibody to its target orto sustain effector functions of an antibody such as CDC and/or ADCC.Preferably, a sequence modified with respect to a specific sequence,when it replaces the specific sequence in an antibody retains binding ofsaid antibody to the target and preferably functions of said antibody asdescribed herein.

Similarly, the teaching given herein with respect to specific antibodiesor hybridomas producing specific antibodies is to be construed so as toalso relate to antibodies characterized by an amino acid sequence and/ornucleic acid sequence which is modified compared to the amino acidsequence and/or nucleic acid sequence of the specific antibodies butbeing functionally equivalent. One important property is to retainbinding of an antibody to its target or to sustain effector functions ofan antibody. Preferably, a sequence modified with respect to a specificsequence, when it replaces the specific sequence in an antibody retainsbinding of said antibody to the target and preferably functions of saidantibody as described herein, e.g. CDC mediated lysis or ADCC mediatedlysis.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR, hypervariable and variable regions can bemodified without losing the ability to bind to a target. For example,CDR regions will be either identical or highly homologous to the regionsof antibodies specified herein. By “highly homologous” it iscontemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or1 or 2 substitutions may be made in the CDRs. In addition, thehypervariable and variable regions may be modified so that they showsubstantial homology with the regions of antibodies specificallydisclosed herein.

It is to be understood that the specific nucleic acids described hereinalso include nucleic acids modified for the sake of optimizing the codonusage in a particular host cell or organism. Differences in codon usageamong organisms can lead to a variety of problems concerningheterologous gene expression. Codon optimization by changing one or morenucleotides of the original sequence can result in an optimization ofthe expression of a nucleic acid, in particular in optimization oftranslation efficacy, in a homologous or heterologous host in which saidnucleic acid is to be expressed.

According to the invention, a variant, derivative, modified form orfragment of a nucleic acid sequence, amino acid sequence, or peptidepreferably has a functional property of the nucleic acid sequence, aminoacid sequence, or peptide, respectively, from which it has been derived.Such functional properties comprise the interaction with or binding toother molecules. In one embodiment, a variant, derivative, modified formor fragment of a nucleic acid sequence, amino acid sequence, or peptideis immunologically equivalent to the nucleic acid sequence, amino acidsequence, or peptide, respectively, from which it has been derived.

Preferably the degree of identity between a specific nucleic acidsequence and a nucleic acid sequence which is modified with respect toor which is a variant of said specific nucleic acid sequence will be atleast 70%, preferably at least 75%, more preferably at least 80%, evenmore preferably at least 90% or most preferably at least 95%, 96%, 97%,98% or 99%. Regarding CLDN6 nucleic acid variants, the degree ofidentity is preferably given for a region of at least about 300, atleast about 400, at least about 450, at least about 500, at least about550, at least about 600 or at least about 630 nucleotides. In preferredembodiments, the degree of identity is given for the entire length ofthe reference nucleic acid sequence, such as the nucleic acid sequencesgiven in the sequence listing. Preferably, the two sequences are capableof hybridizing and forming a stable duplex with one another, withhybridization preferably being carried out under conditions which allowspecific hybridization between polynucleotides (stringent conditions).Stringent conditions are described, for example, in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold SpringHarbor Laboratory press, Cold Spring Harbor, N.Y., 1989 or CurrentProtocols in Molecular Biology, F. M. Ausubel et al., Editors, JohnWiley & Sons, Inc., New York and refer, for example, to hybridization at65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate, pH 7. After hybridization, the membrane to which the DNA hasbeen transferred is washed, for example, in 2×SSC at room temperatureand then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C.

The term “variant” according to the invention also includes mutants,splice variants, conformations, isoforms, allelic variants, speciesvariants and species homologs, in particular those which are naturallypresent. An allelic variant relates to an alteration in the normalsequence of a gene, the significance of which is often unclear. Completegene sequencing often identifies numerous allelic variants for a givengene. A species homolog is a nucleic acid or amino acid sequence with adifferent species of origin from that of a given nucleic acid or aminoacid sequence.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants. Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between aspecific amino acid sequence and an amino acid sequence which ismodified with respect to or which is a variant of said specific aminoacid sequence such as between amino acid sequences showing substantialhomology will be at least 70%, preferably at least 80%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%. The degree of similarity or identity is given preferably for anamino acid region which is at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or about100% of the entire length of the reference amino acid sequence. Forexample, if the reference amino acid sequence consists of 200 aminoacids, the degree of similarity or identity is given preferably for atleast about 20, at least about 40, at least about 60, at least about 80,at least about 100, at least about 120, at least about 140, at leastabout 160, at least about 180, or about 200 amino acids, preferablycontinuous amino acids. Regarding CLDN6 polypeptide variants, the degreeof similarity or identity is given preferably for a region of at leastabout 100, at least about 120, at least about 140, at least about 160,at least about 180, at least about 200, or at least about 210 aminoacids. In preferred embodiments, the degree of similarity or identity isgiven for the entire length of the reference amino acid sequence such asthe amino acid sequences given in the sequence listing. The alignmentfor determining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two polypeptide or nucleicacid sequences indicates the percentage of amino acids or nucleotidesthat are identical between the sequences.

The “percentage identity” is obtained after the best alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly and over their entire length.Sequence comparisons between two nucleotide or amino acid sequences areconventionally carried out by comparing these sequences after havingaligned them optimally, said comparison being carried out by segment orby “window of comparison” in order to identify and compare local regionsof sequence similarity. The optimal alignment of the sequences forcomparison may be produced, besides manually, by means of the localhomology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482,by means of the local homology algorithm of Neddleman and Wunsch, 1970,J. Mol. Biol. 48, 443, by means of the similarity search method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or bymeans of computer programs which use these algorithms (GAP, BESTFIT,FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

“Conservative substitutions,” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Some preferredsubstitutions may be made among the following groups: (i) S and T; (ii)P and G; and (iii) A, V, L and I. Given the known genetic code, andrecombinant and synthetic DNA techniques, the skilled scientist readilycan construct DNAs encoding the conservative amino acid variants.

The present invention comprises antibodies in which alterations havebeen made in the Fc region in order to change the functional orpharmacokinetic properties of the antibodies. Such alterations mayresult in a decrease or increase of C1q binding and CDC or of FcγRbinding and ADCC. Substitutions can, for example, be made in one or moreof the amino acid residues of the heavy chain constant region, therebycausing an alteration in an effector function while retaining theability to bind to the antigen as compared with the modified antibody,cf. U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260.

The in vivo half-life of antibodies can be improved by modifying thesalvage receptor epitope of the Ig constant domain or an Ig-likeconstant domain such that the molecule does not comprise an intact CH2domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022 and U.S.Pat. No. 6,194,551. The in vivo half-life can furthermore be increasedby making mutations in the Fc region, e.g., by substituting threoninefor leucine at position 252, by substituting threonine for serine atposition 254, or by substituting threonine for phenylalanine at position256, cf. U.S. Pat. No. 6,277,375.

Furthermore, the glycosylation pattern of antibodies can be modified inorder to change the effector function of the antibodies. For example,the antibodies can be expressed in a transfectoma which does not add thefucose unit normally attached to Asn at position 297 of the Fc region inorder to enhance the affinity of the Fc region for Fc-Receptors which,in turn, will result in an increased ADCC of the antibodies in thepresence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733.Furthermore, modification of galactosylation can be made in order tomodify CDC.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-CLDN6 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CLDN6antibodies can be screened for binding activity.

According to the invention the term “cell” or “host cell” preferablyrelates to an intact cell, i.e. a cell with an intact membrane that hasnot released its normal intracellular components such as enzymes,organelles, or genetic material. An intact cell preferably is a viablecell, i.e. a living cell capable of carrying out its normal metabolicfunctions. Preferably said term relates according to the invention toany cell which can be transformed or transfected with an exogenousnucleic acid. The term “cell” includes according to the inventionprokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., dendriticcells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELAcells, yeast cells, and insect cells). The exogenous nucleic acid may befound inside the cell (i) freely dispersed as such, (ii) incorporated ina recombinant vector, or (iii) integrated into the host cell genome ormitochondrial DNA. Mammalian cells are particularly preferred, such ascells from humans, mice, hamsters, pigs, goats, and primates. The cellsmay be derived from a large number of tissue types and include primarycells and cell lines. Specific examples include keratinocytes,peripheral blood leukocytes, bone marrow stem cells, and embryonic stemcells. In further embodiments, the cell is an antigen-presenting cell,in particular a dendritic cell, a monocyte, or macrophage. The term“host cell”, as used herein, preferably is intended to refer to a cellinto which a recombinant expression vector has been introduced.

A cell which comprises a nucleic acid molecule preferably express thepeptide or protein encoded by the nucleic acid.

The terms “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably heavy and/or light chaintransgenes, or transchromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is preferably capableof expressing the transgenes. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humananti-CLDN6 antibodies when immunized with CLDN6 antigen and/or cellsexpressing CLDN6. The human heavy chain transgene can be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,e.g., HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chaintransgene can be maintained extrachromosomally, as is the case fortranschromosomal (e.g., KM) mice as described in WO 02/43478. Suchtransgenic and transchromosomal mice may be capable of producingmultiple isotypes of human monoclonal antibodies to CLDN6 (e.g., IgG,IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level, e.g. in the level of proliferation of cells.The term “inhibit” or similar phrases includes a complete or essentiallycomplete inhibition, i.e. a reduction to zero or essentially to zero.

Terms such as “increasing” or “enhancing” preferably relate to anincrease or enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%. These terms may also relate to circumstances, wherein attime zero there is no detectable signal for a certain compound orcondition and at a particular time point later than time zero there is adetectable signal for a certain compound or condition.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. In the context of the present invention,the term “immunologically equivalent” is preferably used with respect tothe immunological effects or properties of a peptide or peptide variantused for immunization. A particular immunological property is theability to bind to antibodies and, where appropriate, generate an immuneresponse, preferably by stimulating the generation of antibodies. Forexample, an amino acid sequence is immunologically equivalent to areference amino acid sequence if said amino acid sequence when exposedto the immune system of a subject induces an immune reaction, preferablyantibodies, having a specificity of reacting with the reference aminoacid sequence, such as the reference amino acid sequence forming part ofCLDN6.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result in the inhibition of tumor growth and/or inhibitionof tumor development, including inhibition of tumor dissemination andmetastasis. Preferably, immune effector functions result in killing oftumor cells. Preferably, the immune effector functions in the context ofthe present invention are antibody-mediated effector functions. Suchfunctions comprise complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), induction ofapoptosis in the cells carrying the tumor-associated antigen, forexample, by binding of the antibody to a surface antigen, and/orinhibition of proliferation of the cells carrying the tumor-associatedantigen, preferably ADCC and/or CDC. Thus, antibodies that are capableof mediating one or more immune effector functions are preferably ableto mediate killing of cells by inducing CDC-mediated lysis,ADCC-mediated lysis, apoptosis, homotypic adhesion, and/or phagocytosis,preferably by inducing CDC-mediated lysis and/or ADCC-mediated lysis.Antibodies may also exert an effect simply by binding totumor-associated antigens on the surface of a tumor cell. For example,antibodies may block the function of the tumor-associated antigen orinduce apoptosis just by binding to the tumor-associated antigen on thesurface of a tumor cell.

DETAILED DESCRIPTION OF THE INVENTION

Mechanisms of mAb Action

Although the following provides considerations regarding the mechanismunderlying the therapeutic efficacy of antibodies of the invention it isnot to be considered as limiting to the invention in any way.

The antibodies described herein may interact with components of theimmune system, preferably through ADCC or CDC. Antibodies of theinvention can also be used to target payloads (e.g., radioisotopes,drugs or toxins) to directly kill tumor cells or can be usedsynergistically with traditional chemotherapeutic agents, attackingtumors through complementary mechanisms of action that may includeanti-tumor immune responses that may have been compromised owing to achemotherapeutic's cytotoxic side effects on T lymphocytes. However,antibodies of the invention may also exert an effect simply by bindingto CLDN6 on the cell surface, thus, e.g. blocking proliferation of thecells.

Antibody-Dependent Cell-Mediated Cytotoxicity

ADCC describes the cell-killing ability of effector cells as describedherein, in particular lymphocytes, which preferably requires the targetcell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cellsand the antibody Fc domains engage Fc receptors (FcR) on the surface ofimmune effector cells. Several families of Fc receptors have beenidentified, and specific cell populations characteristically expressdefined Fc receptors. ADCC can be viewed as a mechanism to directlyinduce a variable degree of immediate tumor destruction that leads toantigen presentation and the induction of tumor-directed T-cellresponses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and host-derived antibody responses.

Complement-Dependent Cytotoxicity

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple C1q binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (C1q is one ofthree subcomponents of complement C1). Preferably these uncloaked C1qbinding sites convert the previously low-affinity C1q-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell.

Production of Antibodies

Antibodies of the invention can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature 256: 495 (1975). Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibodies can be employed, e.g., viral or oncogenic transformation ofB-lymphocytes or phage display techniques using libraries of antibodygenes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodiesdirected against CLDN6 can be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice known as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice.” The production ofhuman antibodies in such transgenic mice can be performed as describedin detail for CD20 in WO2004 035607

Yet another strategy for generating monoclonal antibodies is to directlyisolate genes encoding antibodies from lymphocytes producing antibodiesof defined strategy e.g. see Babcock et al., 1996; A novel strategy forgenerating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined strategy. For details of recombinantantibody engineering see also Welschof and Kraus, Recombinant antibodiesfor cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo AntibodyEngineering ISBN 1-58829-092-1.

Immunizations

To generate antibodies to CLDN6, mice can be immunized withcarrier-conjugated peptides derived from the CLDN6 sequence, an enrichedpreparation of recombinantly expressed CLDN6 antigen or fragmentsthereof and/or cells expressing CLDN6 or fragments thereof, asdescribed. Alternatively, mice can be immunized with DNA encoding fulllength human CLDN6 or fragments thereof. In the event that immunizationsusing a purified or enriched preparation of the CLDN6 antigen do notresult in antibodies, mice can also be immunized with cells expressingCLDN6, e.g., a cell line, to promote immune responses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of anti-CLDN6immunoglobulin can be used for fusions. Mice can be boostedintraperitonealy or intravenously with CLDN6 expressing cells 3-5 daysbefore sacrifice and removal of the spleen to increase the rate ofspecific antibody secreting hybridomas.

Generation of Hybridomas Producing Monoclonal Antibodies

To generate hybridomas producing monoclonal antibodies to CLDN6, cellsfrom lymph nodes or spleens obtained from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can then be screened for theproduction of antigen-specific antibodies. Individual wells can then bescreened by ELISA for antibody secreting hybridomas. ByImmunofluorescence and FACS analysis using CLDN6 expressing cells,antibodies with specificity for CLDN6 can be identified. The antibodysecreting hybridomas can be replated, screened again, and if stillpositive for anti-CLDN6 monoclonal antibodies can be subcloned bylimiting dilution. The stable subclones can then be cultured in vitro togenerate antibody in tissue culture medium for characterization.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as are well known in the art(Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. Afterintroduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Use of Partial Antibody Sequences to Express Intact Antibodies (i.e.Humanization and Chimerisation).

a) Chimerization

Murine monoclonal antibodies can be used as therapeutic antibodies inhumans when labeled with toxins or radioactive isotopes. Nonlabeledmurine antibodies are highly immunogenic in man when repetitivelyapplied leading to reduction of the therapeutic effect. The mainimmunogenicity is mediated by the heavy chain constant regions. Theimmunogenicity of murine antibodies in man can be reduced or completelyavoided if respective antibodies are chimerized or humanized. Chimericantibodies are antibodies, the different portions of which are derivedfrom different animal species, such as those having a variable regionderived from a murine antibody and a human immunoglobulin constantregion. Chimerisation of antibodies is achieved by joining of thevariable regions of the murine antibody heavy and light chain with theconstant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

b) Humanization

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino terminal portion offramework region 1 and in the carboxy-terminal portion of frameworkregion 4. Furthermore, many somatic mutations do not significantly alterthe binding properties of the antibody. For this reason, it is notnecessary to obtain the entire DNA sequence of a particular antibody inorder to recreate an intact recombinant antibody having bindingproperties similar to those of the original antibody (see WO 99/45962).Partial heavy and light chain sequences spanning the CDR regions aretypically sufficient for this purpose. The partial sequence is used todetermine which germline variable and joining gene segments contributedto the recombined antibody variable genes. The germline sequence is thenused to fill in missing portions of the variable regions. Heavy andlight chain leader sequences are cleaved during protein maturation anddo not contribute to the properties of the final antibody. To addmissing sequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences.

The synthetic heavy and kappa chain sequences can differ from thenatural sequences in three ways: strings of repeated nucleotide basesare interrupted to facilitate oligonucleotide synthesis and PCRamplification; optimal translation initiation sites are incorporatedaccording to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and HindIII sites are engineered upstream of thetranslation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotides approximately at the midpoint of thecorresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assembled into overlapping double stranded setsthat span segments of 150-400 nucleotides. The pools are then used astemplates to produce PCR amplification products of 150-400 nucleotides.Typically, a single variable region oligonucleotide set will be brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It may alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs.

The reconstructed chimerized or humanized heavy and light chain variableregions are then combined with cloned promoter, leader, translationinitiation, constant region, 3′ untranslated, polyadenylation, andtranscription termination sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains. Plasmids for use in construction ofexpression vectors for human IgGκ are described. The plasmids can beconstructed so that PCR amplified V heavy and V kappa light chain cDNAsequences can be used to reconstruct complete heavy and light chainminigenes. These plasmids can be used to express completely human, orchimeric IgG1, Kappa or IgG4, Kappa antibodies. Similar plasmids can beconstructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features of theanti-CLDN6 antibodies of the invention, are used to create structurallyrelated humanized anti-CLDN6 antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto CLDN6. More specifically, one or more CDR regions of mouse monoclonalantibodies can be combined recombinantly with known human frameworkregions and CDRs to create additional, recombinantly-engineered,humanized anti-CLDN6 antibodies of the invention.

Binding to Antigen Expressing Cells

The ability of the antibody to bind CLDN6 can be determined usingstandard binding assays, such as those set forth in the examples (e.g.,ELISA, Western Blot, Immunofluorescence and flow cytometric analysis)

Isolation and Characterization of Antibodies

To purify anti-CLDN6 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Alternatively, anti-CLDN6 antibodies can be produced in dialysis basedbioreactors. Supernatants can be filtered and, if necessary,concentrated before affinity chromatography with protein G-sepharose orprotein A-sepharose. Eluted IgG can be checked by gel electrophoresisand high performance liquid chromatography to ensure purity. The buffersolution can be exchanged into PBS, and the concentration can bedetermined by OD280 using 1.43 extinction coefficient. The monoclonalantibodies can be aliquoted and stored at −80° C.

To determine if the selected anti-CLDN6 monoclonal antibodies bind tounique epitopes, site-directed or multi-site directed mutagenesis can beused.

Isotype Determination

To determine the isotype of purified antibodies, isotype ELISAs withvarious commercial kits (e.g. Zymed, Roche Diagnostics) can beperformed. Wells of microtiter plates can be coated with anti-mouse Ig.After blocking, the plates are reacted with monoclonal antibodies orpurified isotype controls, at ambient temperature for two hours. Thewells can then be reacted with either mouse IgG1, IgG2a, IgG2b or IgG3,IgA or mouse IgM-specific peroxidase-conjugated probes. After washing,the plates can be developed with ABTS substrate (1 mg/ml) and analyzedat OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal AntibodyIsotyping Kit (Roche, Cat. No. 1493027) may be used as described by themanufacturer.

Flow Cytometric Analysis

In order to demonstrate presence of anti-CLDN6 antibodies in sera ofimmunized mice or binding of monoclonal antibodies to living cellsexpressing CLDN6, flow cytometry can be used. Cell lines expressingnaturally or after transfection CLDN6 and negative controls lackingCLDN6 expression (grown under standard growth conditions) can be mixedwith various concentrations of monoclonal antibodies in hybridomasupernatants or in PBS containing 1% FBS, and can be incubated at 4° C.for 30 min. After washing, the APC- or Alexa647-labeled anti IgGantibody can bind to CLDN6-bound monoclonal antibody under the sameconditions as the primary antibody staining. The samples can be analyzedby flow cytometry with a FACS instrument using light and side scatterproperties to gate on single, living cells. In order to distinguishCLDN6-specific monoclonal antibodies from non-specific binders in asingle measurement, the method of co-transfection can be employed. Cellstransiently transfected with plasmids encoding CLDN6 and a fluorescentmarker can be stained as described above. Transfected cells can bedetected in a different fluorescence channel than antibody-stainedcells. As the majority of transfected cells express both transgenes,CLDN6-specific monoclonal antibodies bind preferentially to fluorescencemarker expressing cells, whereas non-specific antibodies bind in acomparable ratio to non-transfected cells. An alternative assay usingfluorescence microscopy may be used in addition to or instead of theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence microscopy.

Immunofluorescence Microscopy

In order to demonstrate presence of anti-CLDN6 antibodies in sera ofimmunized mice or binding of monoclonal antibodies to living cellsexpressing CLDN6, immunofluorescence microscopy analysis can be used.For example, cell lines expressing either spontaneously or aftertransfection CLDN6 and negative controls lacking CLDN6 expression aregrown in chamber slides under standard growth conditions in DMEM/F12medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine,100 IU/ml penicillin and 100 μg/ml streptomycin. Cells can then be fixedwith methanol or paraformaldehyde or left untreated. Cells can then bereacted with monoclonal antibodies against CLDN6 for 30 min. at 25° C.After washing, cells can be reacted with an Alexa555-labelled anti-mouseIgG secondary antibody (Molecular Probes) under the same conditions.Cells can then be examined by fluorescence microscopy.

Total CLDN6 levels in cells can be observed when cells are methanolfixed or paraformaldehyde fixed and permeabilized with Triton X-100. Inliving cells and non-permeabilized, paraformaldehyde fixed cells surfacelocalization of CLDN6 can be examined. Additionally targeting of CLDN6to tight junctions can be analyzed by co-staining with tight junctionmarkers such as ZO-1. Furthermore, effects of antibody binding and CLDN6localization within the cell membrane can be examined.

Western Blot

Anti-CLDN6 IgG can be further tested for reactivity with CLDN6 antigenby Western Blotting. Briefly, cell extracts from cells expressing CLDN6and appropriate negative controls can be prepared and subjected tosodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens will be transferred tonitrocellulose membranes, blocked, and probed with the monoclonalantibodies to be tested. IgG binding can be detected using anti-mouseIgG peroxidase and developed with ECL substrate.

Immunohistochemistry

Anti-CLDN6 mouse IgGs can be further tested for reactivity with CLDN6antigen by Immunohistochemistry in a manner well known to the skilledperson, e.g. using paraformaldehyde or acetone fixed cryosections orparaffin embedded tissue sections fixed with paraformaldehyde fromnon-cancer tissue or cancer tissue samples obtained from patients duringroutine surgical procedures or from mice carrying xenografted tumorsinoculated with cell lines expressing spontaneously or aftertransfection CLDN6. For immunostaining, antibodies reactive to CLDN6 canbe incubated followed by horseradish-peroxidase conjugated goatanti-mouse or goat anti-rabbit antibodies (DAKO) according to thevendors instructions.

Phagocytic and Cell Killing Activities of Antibodies In Vitro

In addition to binding specifically to CLDN6, anti-CLDN6 antibodies canbe tested for their ability to mediate phagocytosis and killing of cellsexpressing CLDN6 and being characterized by association of CLDN6 withtheir cell surface. The testing of monoclonal antibody activity in vitrowill provide an initial screening prior to testing in vivo models.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC):

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes,mononuclear cells or other effector cells, from healthy donors can bepurified by Ficoll Hypaque density centrifugation, followed by lysis ofcontaminating erythrocytes. Washed effector cells can be suspended inRPMI supplemented with 10% heat-inactivated fetal calf serum or,alternatively with 5% heat-inactivated human serum and mixed with ⁵¹Crlabeled target cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface, at various ratios ofeffector cells to target cells. Alternatively, the target cells may belabeled with a fluorescence enhancing ligand (BATDA). A highlyfluorescent chelate of Europium with the enhancing ligand which isreleased from dead cells can be measured by a fluorometer. Anotheralternative technique may utilize the transfection of target cells withluciferase. Added lucifer yellow may then be oxidated by viable cellsonly. Purified anti-CLDN6 IgGs can then be added at variousconcentrations. Irrelevant human IgG can be used as negative control.Assays can be carried out for 4 to 20 hours at 37° C. depending on theeffector cell type used. Samples can be assayed for cytolysis bymeasuring ⁵¹Cr release or the presence of the EuTDA chelate in theculture supernatant. Alternatively, luminescence resulting from theoxidation of lucifer yellow can be a measure of viable cells.

Anti-CLDN6 monoclonal antibodies can also be tested in variouscombinations to determine whether cytolysis is enhanced with multiplemonoclonal antibodies.

Complement Dependent Cytotoxicity (CDC):

Monoclonal anti-CLDN6 antibodies can be tested for their ability tomediate CDC using a variety of known techniques. For example, serum forcomplement can be obtained from blood in a manner known to the skilledperson. To determine the CDC activity of mAbs, different methods can beused. ⁵¹Cr release can for example be measured or elevated membranepermeability can be assessed using a propidium iodide (PI) exclusionassay. Briefly, target cells can be washed and 5×10⁵/ml can be incubatedwith various concentrations of mAb for 10-30 min. at room temperature orat 37° C. Serum or plasma can then be added to a final concentration of20% (v/v) and the cells incubated at 37° C. for 20-30 min. All cellsfrom each sample can be added to the PI solution in a FACS tube. Themixture can then be analyzed immediately by flow cytometry analysisusing FACSArray.

In an alternative assay, induction of CDC can be determined on adherentcells. In one embodiment of this assay, cells are seeded 24 h before theassay with a density of 3×10⁴/well in tissue-culture flat-bottommicrotiter plates. The next day growth medium is removed and the cellsare incubated in triplicates with antibodies. Control cells areincubated with growth medium or growth medium containing 0.2% saponinfor the determination of background lysis and maximal lysis,respectively. After incubation for 20 min. at room temperaturesupernatant is removed and 20% (v/v) human plasma or serum in DMEM(prewarmed to 37° C.) is added to the cells and incubated for another 20min. at 37° C. All cells from each sample are added to propidium iodidesolution (10 μg/ml). Then, supernatants are replaced by PBS containing2.5 μg/ml ethidium bromide and fluorescence emission upon excitation at520 nm is measured at 600 nm using a Tecan Safire. The percentagespecific lysis is calculated as follows: % specific lysis=(fluorescencesample-fluorescence background)/(fluorescence maximal lysis-fluorescencebackground)×100.

Inhibition of Cell Proliferation by Monoclonal Antibodies:

To test for the ability to initiate apoptosis, monoclonal anti-CLDN6antibodies can, for example, be incubated with CLDN6 positive tumorcells or CLDN6 transfected tumor cells at 37° C. for about 20 hours. Thecells can be harvested, washed in Annexin-V binding buffer (BDbiosciences), and incubated with Annexin V conjugated with FITC or APC(BD biosciences) for 15 min. in the dark. All cells from each sample canbe added to PI solution (10 μg/ml in PBS) in a FACS tube and assessedimmediately by flow cytometry (as above). Alternatively, a generalinhibition of cell-proliferation by monoclonal antibodies can bedetected with commercially available kits. The DELFIA Cell ProliferationKit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay basedon the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporationduring DNA synthesis of proliferating cells in microplates. IncorporatedBrdU is detected using europium labelled monoclonal antibody. To allowantibody detection, cells are fixed and DNA denatured using Fixsolution. Unbound antibody is washed away and DELFIA inducer is added todissociate europium ions from the labelled antibody into solution, wherethey form highly fluorescent chelates with components of the DELFIAInducer. The fluorescence measured—utilizing time-resolved fluorometryin the detection—is proportional to the DNA synthesis in the cell ofeach well.

Preclinical Studies

Monoclonal antibodies which bind to CLDN6 also can be tested in an invivo model (e.g. in immune deficient mice carrying xenografted tumorsinoculated with cell lines expressing CLDN6, possibly aftertransfection) to determine their efficacy in controlling growth ofCLDN6-expressing tumor cells.

In vivo studies after xenografting CLDN6 expressing tumor cells intoimmunocompromised mice or other animals can be performed usingantibodies of the invention. Antibodies can be adminstered to tumor freemice followed by injection of tumor cells to measure the effects of theantibodies to prevent formation of tumors or tumor-related symptoms.Antibodies can be adminstered to tumor-bearing mice to determine thetherapeutic efficacy of respective antibodies to reduce tumor growth,metastasis or tumor related symptoms. Antibody application can becombined with application of other substances as cystostatic drugs,growth factor inhibitors, cell cycle blockers, angiogenesis inhibitorsor other antibodies to determine synergistic efficacy and potentialtoxicity of combinations. To analyze toxic side effects mediated byantibodies of the invention animals can be inoculated with antibodies orcontrol reagents and thoroughly investigated for symptoms possiblyrelated to CLDN6-antibody therapy. Possible side effects of in vivoapplication of CLDN6 antibodies particularly include toxicity at CLDN6expressing tissues including placenta. Antibodies recognizing CLDN6 inhuman and in other species, e.g. mice, are particularly useful topredict potential side effects mediated by application of monoclonalCLDN6 antibodies in humans.

Epitope Mapping

Mapping of epitopes recognized by antibodies of invention can beperformed as described in detail in “Epitope Mapping Protocols (Methodsin Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in“Epitope Mapping: A Practical Approach” Practical Approach Series, 248by Olwyn M. R. Westwood, Frank C. Hay.

I. Bispecific/Multispecific Molecules which Bind to CLDN6

In yet another embodiment of the invention, antibodies to CLDN6 can bederivatized or linked to another functional molecule, e.g., anotherpeptide or protein (e.g., an Fab′ fragment) to generate a bispecific ormultispecific molecule which binds to multiple binding sites or targetepitopes. For example, an antibody of the invention can be functionallylinked (e.g. by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, peptide or binding mimetic.

Accordingly, the present invention includes bispecific and multispecificmolecules comprising at least one first binding specificity for CLDN6and a second binding specificity for a second target epitope. In aparticular embodiment of the invention, the second target epitope is anFc receptor, e.g. human Fc-gammaRI (CD64) or a human Fc-alpha receptor(CD89), or a T cell receptor, e.g. CD3. Therefore, the inventionincludes bispecific and multispecific molecules capable of binding bothto Fc-gammaR, Fc-alphaR or Fc-epsilonR expressing effector cells (e.g.monocytes, macrophagesor polymorphonuclear cells (PMNs)), and to targetcells expressing CLDN6 and being characterized by association of CLDN6with their cell surface. These bispecific and multispecific moleculesmay target cells expressing CLDN6 and being characterized by associationof CLDN6 with their cell surface to effector cells and may trigger Fcreceptor-mediated effector cell activities, such as phagocytosis ofcells expressing CLDN6 and being characterized by association of CLDN6with their cell surface, antibody dependent cellular cytotoxicity(ADCC), cytokine release, or generation of superoxide anion.

Bispecific and multispecific molecules of the invention can furtherinclude a third binding specificity, in addition to an anti-Fc bindingspecificity and an anti-CLDN6 binding specificity. In one embodiment,the third binding specificity is an anti-enhancement factor (EF)portion, e.g. a molecule which binds to a surface protein involved incytotoxic activity and thereby increases the immune response against thetarget cell. The “anti-enhancement factor portion” can be an antibody,functional antibody fragment or a ligand that binds to a given molecule,e.g., an antigen or a receptor, and thereby results in an enhancement ofthe effect of the binding determinants for the Fc receptor or targetcell antigen. The “anti-enhancement factor portion” can bind an Fcreceptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T cell (e.g.,via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific and multispecific molecules of theinvention comprise as a binding specificity at least one antibody,including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv. Theantibody may also be a light chain or heavy chain dimer, or any minimalfragment thereof such as a Fv or a single chain construct as describedin Ladner et al., U.S. Pat. No. 4,946,778. The antibody may also be abinding-domain immunoglobulin fusion protein as disclosed inUS2003/0118592 and US 2003/0133939.

In one embodiment bispecific and multispecific molecules of theinvention comprise a binding specificity for an Fc-gammaR or anFc-alphaR present on the surface of an effector cell, and a secondbinding specificity for a target cell antigen, e.g., CLDN6.

In one embodiment, the binding specificity for an Fc receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight gamma-chain genes located on chromosome 1.These genes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fc-gamma receptor classes:Fc-gammaRI (CD64), Fc-gammaRII (CD32), and Fc-gammaRIII (CD16). In onepreferred embodiment, the Fc-gamma receptor is a human high affinityFc-gammaRI.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (Fc-alphaRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one alpha-gene(Fc-alphaRI) located on chromosome 19. This gene is known to encodeseveral alternatively spliced transmembrane isoforms of 55 to 110 kDa.Fc-alphaRI (CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. Fc-alphaRI has medium affinity for both IgA1 and IgA2,which is increased upon exposure to cytokines such as G-CSF or GM-CSF(Morton, H. C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four Fc-alphaRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind Fc-alphaRI outside the IgA ligandbinding domain, have been described (Monteiro, R. C. et al. (1992) J.Immunol. 148: 1764).

In another embodiment the bispecific molecule is comprised of twomonoclonal antibodies according to the invention which havecomplementary functional activities, such as one antibody predominatelyworking by inducing CDC and the other antibody predominately working byinducing apoptosis.

An “effector cell specific antibody” as used herein refers to anantibody or functional antibody fragment that binds the Fc receptor ofeffector cells. Preferred antibodies for use in the subject inventionbind the Fc receptor of effector cells at a site which is not bound byendogenous immunoglobulin.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include cells of myeloid or lymphoid origin, e.g,lymphocytes (e.g., B cells and T cells including cytolytic T cells(CTLs), killer cells, natural killer cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and basophils. Some effector cells express specific Fc receptorsand carry out specific immune functions. In preferred embodiments, aneffector cell is capable of inducing antibody-dependent cellularcytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. Forexample, monocytes, macrophages, which express FcR are involved inspecific killing of target cells and presenting antigens to othercomponents of the immune system, or binding to cells that presentantigens. In other embodiments, an effector cell can phagocytose atarget antigen, target cell, or microorganism. The expression of aparticular FcR on an effector cell can be regulated by humoral factorssuch as cytokines. For example, expression of Fc-gammaRI has been foundto be up-regulated by interferon gamma (IFN-γ). This enhanced expressionincreases the cytotoxic activity of Fc-gammaRI-bearing cells againsttargets. An effector cell can phagocytose or lyse a target antigen or atarget cell.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by an antibody of the invention.In preferred embodiments, the target cell is a cell expressing oroverexpressing CLDN6 and being characterized by association of CLDN6with its cell surface. Cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface typically include tumorcells.

II. Immunoconjugates

In another aspect, the present invention features an anti-CLDN6 antibodyconjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug(e.g., an immunosuppressant) or a radioisotope. Such conjugates arereferred to herein as “immunoconjugates”. Immunoconjugates which includeone or more cytotoxins are referred to as “immunotoxins”. A cytotoxin orcytotoxic agent includes any agent that is detrimental to and, inparticular, kills cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Suitable therapeutic agents for forming immunoconjugates of theinvention include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents(e.g., vincristine and vinblastine). In a preferred embodiment, thetherapeutic agent is a cytotoxic agent or a radiotoxic agent. In anotherembodiment, the therapeutic agent is an immunosuppressant. In yetanother embodiment, the therapeutic agent is GM-CSF. In a preferredembodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin,sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies of the present invention also can be conjugated to aradioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generatecytotoxic radiopharmaceuticals for treating a CLDN6-related disorder,such as a cancer. The antibody conjugates of the invention can be usedto modify a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-γ; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62: 119-58 (1982).

In a further embodiment, the antibodies according to the invention areattached to a linker-chelator, e.g., tiuxetan, which allows for theantibody to be conjugated to a radioisotope.

III. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofantibodies of the present invention. The pharmaceutical compositions maybe formulated with pharmaceutically acceptable carriers or diluents aswell as any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995. In one embodiment, the compositionsinclude a combination of multiple (e.g., two or more) isolatedantibodies of the invention which act by different mechanisms, e.g., oneantibody which predominately acts by inducing CDC in combination withanother antibody which predominately acts by inducing apoptosis.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one anti-inflammatory agent or at least oneimmunosuppressive agent. In one embodiment such therapeutic agentsinclude one or more anti-inflammatory agents, such as a steroidal drugor a NSAID (nonsteroidal anti-inflammatory drug). Preferred agentsinclude, for example, aspirin and other salicylates, Cox-2 inhibitors,such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such asibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn),sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene),ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen),etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

In another embodiment, such therapeutic agents include agents leading tothe depletion or functional inactivation of regulatory T cells like lowdose cyclophosphamid, anti-CTLA4 antibodies, anti-IL2 oranti-IL2-receptor antibodies.

In yet another embodiment, such therapeutic agents include one or morechemotherapeutics, such as Taxol derivatives, taxotere, gemcitabin,5-Fluoruracil, doxorubicin (Adriamycin), cisplatin (Platinol),cyclophosphamide (Cytoxan, Procytox, Neosar). In another embodiment,antibodies of the present invention may be administered in combinationwith chemotherapeutic agents, which preferably show therapeutic efficacyin patients suffering from cancer, e.g. cancer types as describedherein.

In yet another embodiment, the antibodies of the invention may beadministered in conjunction with radiotherapy and/or autologousperipheral stem cell or bone marrow transplantation.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, e.g., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66: 1-19).

Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and infrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe presence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

In one embodiment, the antibodies of the invention may be administeredby infusion, preferably slow continuous infusion over a long period,such as more than 24 hours, in order to reduce toxic side effects. Theadministration may also be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage can be determined or adjusted bymeasuring the amount of circulating monoclonal anti-CLDN6 antibodiesupon administration in a biological sample by using anti-idiotypicantibodies which target the anti-CLDN6 antibodies.

In yet another embodiment, the antibodies are administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In still another embodiment, the antibodies according to the inventionmay be administered by a regimen including one infusion of an antibodyagainst CLDN6 followed by an infusion of an antibody against CLDN6conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9days later.

In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes. In a more preferred embodiment,the liposomes include a targeting moiety. In a most preferredembodiment, the therapeutic compounds in the liposomes are delivered bybolus injection to a site proximal to the desired area, e.g., the siteof a tumor. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

In a further embodiment, antibodies of the invention can be formulatedto prevent or reduce their transport across the placenta. This can bedone by methods known in the art, e.g., by PEGylation of the antibodiesor by use of F(ab)₂′ fragments. Further references can be made to“Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biologicalactivities of polyethylene-glycol immunoglobulin conjugates. Resistanceto enzymatic degradation. J. Immunol. Methods, 152: 177-190; and to“Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann.Allergy Asthma Immunol. 74: 279-283.

A “therapeutically effective dosage” for tumor therapy can be measuredby objective tumor responses which can either be complete or partial. Acomplete response (CR) is defined as no clinical, radiological or otherevidence of disease. A partial response (PR) results from a reduction inaggregate tumor size of greater than 50%. Median time to progression isa measure that characterizes the durability of the objective tumorresponse.

A “therapeutically effective dosage” for tumor therapy can also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer can be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition can be evaluated by examining the ability ofthe compound to inhibit cell growth or apoptosis by in vitro assaysknown to the skilled practitioner. A therapeutically effective amount ofa therapeutic compound can decrease tumor size, or otherwise amelioratesymptoms in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

The antibodies (including immunoconjugates, bispecifics/multispecifics,compositions and other derivatives described herein) of the presentinvention have numerous therapeutic utilities involving the treatment ofdisorders involving cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface. For example, theantibodies can be administered to cells in culture, e.g., in vitro or exvivo, or to human subjects, e.g., in vivo, to treat or prevent a varietyof disorders such as those described herein. Preferred subjects includehuman patients having disorders that can be corrected or ameliorated, bykilling diseased cells, in particular cells characterized by an alteredexpression pattern of CLDN6 and/or an altered pattern of association ofCLDN6 with their cell surface compared to normal cells.

For example, in one embodiment, antibodies of the present invention canbe used to treat a subject with a tumorigenic disorder, e.g., a disordercharacterized by the presence of tumor cells expressing CLDN6 and beingcharacterized by association of CLDN6 with their cell surface. Examplesof tumorigenic diseases which can be treated and/or prevented encompassall CLDN6 expressing cancers and tumor entities including thosedescribed herein.

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to prevent a disease described herein.

In another embodiment, antibodies of the invention can be used to detectlevels of CLDN6 or particular forms of CLDN6, or levels of cells whichcontain CLDN6 on their membrane surface, which levels can then be linkedto certain diseases or disease symptoms such as described above.Alternatively, the antibodies can be used to deplete or interact withthe function of cells expressing CLDN6 and being characterized byassociation of CLDN6 with their cell surface, thereby implicating thesecells as important mediators of the disease. This can be achieved bycontacting a sample and a control sample with the anti-CLDN6 antibodyunder conditions that allow for the formation of a complex between theantibody and CLDN6. Any complexes formed between the antibody and CLDN6are detected and compared in the sample and a control sample, i.e. areference sample.

Antibodies of the invention can be initially tested for their bindingactivity associated with therapeutic or diagnostic uses in vitro. Forexample, the antibodies can be tested using flow cytometric assays asdescribed herein.

The antibodies of the invention can be used to elicit in vivo or invitro one or more of the following biological activities: to inhibit thegrowth of and/or differentiation of a cell expressing CLDN6 and beingcharacterized by association of CLDN6 with its cell surface; to kill acell expressing CLDN6 and being characterized by association of CLDN6with its cell surface; to mediate phagocytosis or ADCC of a cellexpressing CLDN6 and being characterized by association of CLDN6 withits cell surface in the presence of effector cells; to mediate CDC of acell expressing CLDN6 and being characterized by association of CLDN6with its cell surface in the presence of complement; to mediateapoptosis of a cell expressing CLDN6 and being characterized byassociation of CLDN6 with its cell surface; to induce homotypicadhesion; and/or to induce translocation into lipid rafts upon bindingCLDN6.

In a particular embodiment, the antibodies are used in vivo or in vitroto treat, prevent or diagnose a variety of CLDN6-related diseases.Examples of CLDN6-related diseases include, among others, cancers suchas those described herein.

As described above, anti-CLDN6 antibodies of the invention can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent, antiangiogeneic agent or andimmunosuppressive agent to reduce the induction of immune responsesagainst the antibodies of invention. The antibody can be linked to theagent (as an immunocomplex) or can be administered separate from theagent. In the latter case (separate administration), the antibody can beadministered before, after or concurrently with the agent or can beco-administered with other known therapies, e.g., an anti-cancertherapy, e.g., radiation. Such therapeutic agents include, among others,anti-neoplastic agents such as listed above. Co-administration of theanti-CLDN6 antibodies of the present invention with chemotherapeuticagents provides two anti-cancer agents which operate via differentmechanisms yielding a cytotoxic effect to tumor cells. Suchco-administration can solve problems due to development of resistance todrugs or a change in the antigenicity of the tumor cells which wouldrender them unreactive with the antibody.

The compositions (e.g., antibodies, multispecific and bispecificmolecules and immunoconjugates) of the invention which have complementbinding sites, such as portions from IgG1, -2, or -3 or IgM which bindcomplement, can also be used in the presence of complement. In oneembodiment, ex vivo treatment of a population of cells comprising targetcells with a binding agent of the invention and appropriate effectorcells can be supplemented by the addition of complement or serumcontaining complement. Phagocytosis of target cells coated with abinding agent of the invention can be improved by binding of complementproteins. In another embodiment target cells coated with thecompositions of the invention can also be lysed by complement. In yetanother embodiment, the compositions of the invention do not activatecomplement.

The compositions of the invention can also be administered together withcomplement. Accordingly, within the scope of the invention arecompositions comprising antibodies, multispecific or bispecificmolecules and serum or complement. These compositions are advantageousin that the complement is located in close proximity to the antibodies,multispecific or bispecific molecules.

Alternatively, the antibodies, multispecific or bispecific molecules ofthe invention and the complement or serum can be administeredseparately. Binding of the compositions of the present invention totarget cells may cause translocation of the CLDN6 antigen-antibodycomplex into lipid rafts of the cell membrane. Such translocationcreates a high density of antigen-antibody complexes which mayefficiently activate and/or enhance CDC.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., antibodies andimmunoconjugates) and instructions for use. The kit can further containone or more additional reagents, such as an immunosuppressive reagent, acytotoxic agent or a radiotoxic agent, or one or more additionalantibodies of the invention (e.g., an antibody having a complementaryactivity).

Accordingly, patients treated with antibody compositions of theinvention can be additionally administered (prior to, simultaneouslywith, or following administration of a antibody of the invention) withanother therapeutic agent, such as a cytotoxic or radiotoxic agent,which enhances or augments the therapeutic effect of the antibodies ofthe invention.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fc-gamma or Fc-alpha receptors by, for example, treating thesubject with a cytokine. Preferred cytokines include granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF). Other important agents for increasing thetherapeutic efficacy of the antibodies and pharmaceutical compositionsdescribed herein are β-glucans which are homopolysaccharides of branchedglucose residues and are produced by a variety of plants andmicroorganisms, for example, bacteria, algae, fungi, yeast and grains.Fragments of β-glucans produced by organisms may be also be used.Preferably, the β-glucan is a polymer of β(1,3) glucose wherein at leastsome of the backbone glucose units, e.g. 3-6% of the backbone glucoseunits, possess branches such as β(1,6) branches.

In a particular embodiment, the invention provides methods for detectingthe presence of CLDN6 antigen in a sample, or measuring the amount ofCLDN6 antigen, comprising contacting the sample, and a control sample,with an antibody which specifically binds to CLDN6, under conditionsthat allow for formation of a complex between the antibody or portionthereof and CLDN6. The formation of a complex is then detected, whereina difference complex formation between the sample compared to thecontrol sample is indicative for the presence of CLDN6 antigen in thesample.

In still another embodiment, the invention provides a method fordetecting the presence or quantifying the amount of cells expressingCLDN6 and being characterized by association of CLDN6 with their cellsurface in vivo or in vitro. The method comprises (i) administering to asubject a composition of the invention conjugated to a detectablemarker; and (ii) exposing the subject to a means for detecting saiddetectable marker to identify areas containing cells expressing CLDN6and being characterized by association of CLDN6 with their cell surface.

Methods as described above are useful, in particular, for diagnosingCLDN6-related diseases and/or the localization of CLDN6-related diseasessuch as cancer diseases. Preferably an amount of CLDN6 in a sample whichis higher than the amount of CLDN6 in a control sample is indicative forthe presence of a CLDN6-related disease in a subject, in particular ahuman, from which the sample is derived.

When used in methods as described above, an antibody described hereinmay be provided with a label that functions to: (i) provide a detectablesignal; (ii) interact with a second label to modify the detectablesignal provided by the first or second label, e.g. FRET (FluorescenceResonance Energy Transfer); (iii) affect mobility, e.g. electrophoreticmobility, by charge, hydrophobicity, shape, or other physicalparameters, or (iv) provide a capture moiety, e.g., affinity,antibody/antigen, or ionic complexation. Suitable as label arestructures, such as fluorescent labels, luminescent labels, chromophorelabels, radioisotopic labels, isotopic labels, preferably stableisotopic labels, isobaric labels, enzyme labels, particle labels, inparticular metal particle labels, magnetic particle labels, polymerparticle labels, small organic molecules such as biotin, ligands ofreceptors or binding molecules such as cell adhesion proteins orlectins, label-sequences comprising nucleic acids and/or amino acidresidues which can be detected by use of binding agents, etc. Labelscomprise, in a nonlimiting manner, barium sulfate, iocetamic acid,iopanoic acid, calcium ipodate, sodium diatrizoate, megluminediatrizoate, metrizamide, sodium tyropanoate and radio diagnostic,including positron emitters such as fluorine-18 and carbon-11, gammaemitters such as iodine-123, technetium-99m, iodine-131 and indium-111,nuclides for nuclear magnetic resonance, such as fluorine andgadolinium.

In yet another embodiment immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have CLDN6associated with their surface by linking such compounds to the antibody.Thus, the invention also provides methods for localizing ex vivo or invitro cells expressing CLDN6 and being characterized by association ofCLDN6 with their cell surface, such as circulating tumor cells.

The present invention is further illustrated by the following exampleswhich are not be construed as limiting the scope of the invention.

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methodsincluding the use of kits and reagents are carried out according to themanufacturer's information unless specifically indicated.

Example 1 Quantification of CLDN6 Expression in Normal Tissues,Cancerous Tissues and Cell Lines Using Real-Time RT-PCR

Total cellular RNA was extracted from frozen tissue specimens and cancercell lines using RNeasy Mini Kit (Qiagen), primed with a dT₁₈oligonucleotide and reverse-transcribed with Superscript II(GIBCO/Lifetech) according to the manufacturer's instructions. Integrityof the obtained cDNA was tested by amplification of p53 transcripts in a30 cycle PCR. After normalization to HPRT expression of CLDN6 wasquantified using ΔΔCT calculation.

Tissues from three individuals were tested for each normal tissue type.Only trace amounts of CLDN6 transcripts could be detected in normaltissues after 40 cycles of RT-PCR. The only normal tissue slightlyexceeding the expression cutoff was placenta.

In contrast to normal tissues, we found high expression of CLDN6 insamples from ovarian cancer (adenocarcinomas), lung cancer (NSCLC, withhighest frequency and expression levels in adenocarcinomas), gastriccancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer(basal cell carcinoma and squamous cell carcinoma), malignant melanoma,head and neck cancer (malignant pleomorphic adenoma), sarcoma (synovialsarcoma and carcinosarcoma), bile duct cancer, renal cell cancer (clearcell carcinoma and papillary carcinoma), uterine cancer and cancer celllines A2780 (ovarian cancer), NIH-OVCAR3 (ovarian cancer), HCT-116(colon cancer), EFO-27 (ovarian cancer), CPC-N(SCLC), NCI-H552 (NSCLC),SNU-1 (gastric cancer), KATOIII (gastric cancer), YAPC (pancreaticcancer), AGS (gastric cancer), FU97 (gastric cancer), MKN7 (gastriccancer).

Example 2 Quantification of CLDN6 Expression in Normal Tissues,Cancerous Tissues and Cell Lines Using Western Blot Analysis

For Western blot analysis 20 μg of total protein extracted from cellslyzed with Laemmli-lysis buffer was used. Extracts were diluted inreducing sample buffer (Roth), subjected to SDS-PAGE and subsequentlyelectrotransferred onto PVDF membrane (Pall). Immunostaining wasperformed with polyclonal antibodies reactive to CLDN6 (ARP) andbeta-Actin (Abcam) followed by detection of primary antibodies withhorseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbitsecondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normaltissue type. No CLDN6 protein expression was detected in any of thenormal tissues analyzed. In contrast to normal tissues, high expressionof CLDN6 protein was detected in samples from ovarian cancer and lungcancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer),MKN7 (gastric cancer), AGS (gastric cancer), CPC-N(SCLC), HCT-116 (coloncancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma),JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO(placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

Example 3 Immunohistochemical (IHC) Analysis of CLDN6 Expression inNormal Tissues and Cancerous Tissues

Paraffin-embedded tissue sections (4 μm) were incubated for 1 hour at58° C. on a heating plate (HI 1220, Leica). Paraffin was removed fromthe sections by incubating the slides in Roticlear (Roth) for 2×10 minat RT. Afterwards the sections were rehydrated in graded alcohol (99%,2×96%, 80% and 70%, 5 min each). Antigen retrieval was performed byboiling slides at 120° C. (15 psi) for 15 min in 10 mM citrate buffer(pH 6.0)+0.05% Tween-20. Directly after boiling slides were incubated inPBS for 5 min. Endogenous peroxidase activity was blocked with 0.3%hydrogen peroxide in MeOH for 15 min at RT. To avoid non-specificbinding the slides were blocked with 10% goat serum in PBS for 30 min atRT. Thereafter, the slides were incubated with CLDN6-specific polyclonalantibody (1 μg/ml) (ARP) overnight at 4° C. On the next day the slideswere washed with PBS at RT (3×5 min) and incubated with 100 μl of thesecondary antibodies (PowerVision poly HRP-Anti-Rabbit IgG ready-to-use(ImmunoLogic)) for one hour at RT. Afterwards, slides were washed withPBS at RT (3×5 min). Final staining was performed by using the VECTORNovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame).Sections were counterstained with haematoxylin for 90 sec at RT. Afterdehydration with graded alcohol (70%, 80%, 2×96% and 99%, 5 min each)and 10 min incubation in xylol slides were mounted with X-tra Kit(Medite Histotechnic).

No CLDN6 protein expression was detectable in normal tissues from lung,ovary, stomach, colon, pancreas, liver, duodenum or kidney. In contrastto normal tissues, strong or at least significant staining was observedon tissue sections from ovarian cancer, lung cancer, skin cancer,pancreatic cancer, gastric cancer, breast cancer, urinary bladder cancer(transitional cell carcinoma), cervical cancer, testicular cancer(seminoma) and uterine cancer. Staining was clearly accentuated at theplasma membrane of the malignant epithelial cell populations, whereasadjacent stromal and non-malignant epithelial cells were negative. Theseresults indicate that CLDN6 protein is localized at the plasma membraneof malignant cells.

Example 4 Generation of Murine Antibodies Against CLDN6

a. Generation of Expression Vectors Encoding Full Length CLDN6 and CLDN6Fragments

A non-natural, codon-optimized DNA sequence (SEQ ID NO: 3) encoding fulllength CLDN6 (NCBI accession number NP_067018.2, SEQ ID NO: 2) wasprepared by chemical synthesis (GENEART AG, Germany) and cloned into thepcDNA3.1/myc-His vector (Invitrogen, USA) yielding the vector p3953.Insertion of a stop codon allowed the expression of CLDN6 proteinwithout being fused to the vector encoded myc-His tag. Expression ofCLDN6 was tested by Western blot, flow cytometry and immunofluorescenceanalyzes using commercially available anti-CLDN6 antibodies (ARP,01-8865; R&D Systems, MAB3656).

In addition, a codon-optimized DNA sequence (SEQ ID NO: 4) coding forthe putative extracellular domain 2 (EC2) fragment of CLDN6 (SEQ ID NO:6) as a fusion with an N-terminal Ig kappa leader derived signal peptidefollowed by 4 additional amino acids to ensure a correct signalpeptidase cleavage site (SEQ ID NO: 5) was prepared and cloned into thepcDNA3.1/myc-His vector yielding the vector p3974. Prior toimmunization, expression of the EC2 fragment was confirmed byimmunofluorescence microscopy on transiently transfected andparaformaldehyde (PFA)-fixed CHO-K1 cells using a commercially availableanti-myc antibody (Cell Signaling, MAB 2276).

b. Generation of Cell Lines Stably Expressing CLDN6

HEK293 and P3X63Ag8U.1 cell lines stably expressing CLDN6 were generatedby standard techniques using the vector p3953.

c. Immunizations

Balb/c mice were immunized with 25 μg of p3974 plasmid DNA together with4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man from PolyPlusTransfection) (150 mM PEI-Man in H₂O with 5% glucose) by intraperitonealinjection on days 0, 16 and 36. On days 48 and 62 mice were immunized byintraperitoneal injection with P3X63Ag8U.1 myeloma cells transfectedwith p3953 vector to stably express CLDN6. The cells administered on day62 had been irradiated with 3000 rad prior to injection. The presence ofantibodies directed against CLDN6 in sera of mice was monitored byimmunofluorescence microscopy between days 20 and 70 using CHO-K1 cellsco-transfected with nucleic acids encoding CLDN6 and GFP. To this end,24 h following transfection, PFA-fixed or non-fixed cells were incubatedwith a 1:100 dilution of sera from immunized mice for 45 min at roomtemperature (RT). Cells were washed, incubated with an Alexa555-labeledanti-mouse Ig antibody (Molecular Probes) and subjected to fluorescencemicroscopy.

Anti-CLDN6 specific antibodies were detected in serum samples obtainedfrom a mouse on the basis of which the hybridoma F3-6C3-H8 was produced;see FIG. 2.

For generation of monoclonal antibodies, mice with detectable anti-CLDN6immune responses were boosted four days prior to splenectomy byintraperitonal injection of 2×10⁷ HEK293 cells stably transfected withp3953 vector.

d. Generation of Hybridomas Producing Murine Monoclonal AntibodiesAgainst CLDN6

6×10⁷ splenocytes isolated from an immunized mouse were fused with 3×10⁷cells of the mouse myeloma cell line P3X63Ag8.653 (ATCC, CRL 1580) usingPEG 1500 (Roche, CRL 10783641001). Cells were seeded at approximately5×10⁴ cells per well in flat bottom microtiter plates and cultivated forabout two weeks in RPMI selective medium containing 10% heat inactivatedfetal bovine serum, 1% hybridoma fusion and cloning supplement (HFCS,Roche, CRL 11363735), 10 mM HEPES, 1 mM sodium pyruvate, 4.5% glucose,0.1 mM 2-mercaptoethanol, 1×penicillin/streptomycin and 1×HAT supplement(Invitrogen, CRL 21060). After 10 to 14 days, individual wells werescreened by flow cytometry for anti-CLDN6 monoclonal antibodies.Antibody secreting hybridomas were subcloned by limiting dilution andagain tested for anti-CLDN6 monoclonal antibodies. The stable subcloneswere cultured to generate small amounts of antibody in tissue culturemedium for characterization. At least one clone from each hybridomawhich retained the reactivity of the parent cells (tested by flowcytometry) was selected. Nine-vial-cell banks were generated for eachclone and stored in liquid nitrogen.

Example 5 Binding Characteristics of Hybridoma Supernatants andMonoclonal Antibodies

a. Quality Control of Transiently Transfected HEK293T Cells by (i)Western Blot and (ii) Flow Cytometry Analyzes

(i) HEK293T cells were transfected with nucleic acids encoding CLDN3,CLDN4, CLDN6, and CLDN9, respectively, or mock-transfected. Expressionof CLDN3, CLDN4, CLDN6 or CLDN9 in HEK293T cells was determined byWestern blotting. To this end, cells were harvested 24 hours posttransfection and subjected to lysis. The lysate was subjected toSDS-PAGE, blotted onto nitrocellulose membrane and stained withanti-CLDN3(A) (Invitrogen, 34-1700), anti-CLDN4(A) (Zymed, 32-9400),anti-CDLN6(A) (ARP, 01-8865) or anti-CLDN9(A) (Santa Cruz, sc-17672)antibodies which specifically bind to the C-terminus of thecorresponding claudin under denaturing conditions. Following incubationwith a peroxidase-labeled secondary antibody and developing with ECLreagent, a LAS-3000 imager (Fuji) was used for visualization. Bands ofthe expected molecular weights of CLDN3, CLDN4, CLDN6 and CLDN9,respectively, were observed only in the transfected cells but not in thecontrol cells (FIG. 3) demonstrating that HEK293T cells do notendogenously express any of the claudins investigated and thus, are asuitable tool for determining the cross reactivity of CLDN6 antibodies.

(ii) The HEK293T cells of (i) were further analyzed by flow cytometryusing anti-CLDN antibodies recognizing native epitopes (mouse anti-CLDN3IgG2a (R&D, MAB4620), mouse anti-CLDN4 IgG2a (R&D, MAB4219), mouseanti-CLDN6 IgG2b (R&D, MAB3656)). The antibodies obtainable from Sigmaunder the product numbers M9144 and M8894 served as isotype controls.Specificity of these anti-CLDN antibodies was analyzed using HEK293Tcells transiently transfected with nucleic acids encoding CLDN3, CLDN4,CLDN6, and CLDN9, respectively. The anti-CLDN6 antibody showscross-reactivity with CLDN3, CLDN4 and CLDN9. The anti-CLDN4 antibodyshows cross-reactivity with CLDN3, CLDN6 and CLDN9. The anti-CLDN3antibody binds specifically to CLDN3 (FIG. 4).

b. Determination of the Specificity of Monoclonal Antibodies ProducedAccording to the Invention Using Flow Cytometry

HEK293T cells were co-transfected with a vector encoding different CLDNproteins and a vector encoding a fluorescence marker. 24 h posttransfection cells were harvested using 0.05% trypsin/EDTA solution andwashed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide).Cells were transferred into U-bottom microtiter plates at 2×10⁵ cellsper well and incubated for 60 min at 4° C. with hybridoma supernatants.Following washing three times with FACS buffer, cells were incubatedwith an allophycocyanin (APC)-conjugated anti-mouse IgG 1+2a+2b+3specific secondary antibody (Dianova, 115-135-164). Thereafter, cellswere washed twice and binding was assessed by flow cytometry using a BDFACSArray (FIG. 5). The expression of the fluorescence marker is plottedon the horizontal axis against the antibody binding on the verticalaxis. A commercially available mouse anti-CLDN6 IgG2b antibody (R&D,MAB3656) served as a positive control and the antibody obtainable fromSigma under the product number M8894 served as an isotype control.

Antibodies in the supernatants from the monoclonal hybridoma subclonesF3-6C3-H2, F3-6C3-H8, F3-6C3-H9, F3-6C3-D8 and F3-6C3-G4, all derivedfrom hybridoma F3-6C3, were specific for CLDN6 and did not bind toCLDN9, CLDN3 and CLDN4. FIG. 5A exemplarily shows the results for themonoclonal hybridoma subclone F3-6C3-H8. Antibodies in the supernatantfrom the monoclonal hybridoma subclone F3-6C3-H8 also bind to cellstransfected with the (I143V)-SNP variant of CLDN6. Antibodies in thesupernatant from the monoclonal hybridoma subclone F4-4F7-F2 bind toboth CLDN6 and CLDN9 (FIG. 5A). Antibodies in the supernatant from themonoclonal hybridoma subclone F3-7B3-B4 bind to CLDN6, CLDN3 and CLDN9(FIG. 5B). Antibodies in the supernatant from the monoclonal hybridomasubclone F3-3F7-A5 bind to CLDN6, CLDN4 and CLDN9 (FIG. 5B).

Example 6 Generation and Testing of Monoclonal Antibodies Against CLDN6

a. Generation of Expression Vectors Encoding the Extracellular Domain 1of CLDN6

A codon-optimized DNA sequence (SEQ ID NO: 12) coding for the putativeextracellular domain 1 (EC1) fragment of CLDN6 (SEQ ID NO: 7) as afusion with an N-terminal Ig kappa leader derived signal peptidefollowed by 4 additional amino acids to ensure a correct signalpeptidase cleavage site (SEQ ID NO: 13) was prepared and cloned into thepcDNA3.1/myc-His vector yielding the vector p3973. Prior toimmunization, expression of the EC1 fragment was confirmed byimmunofluorescence microscopy on transiently transfected andparaformaldehyde (PFA)-fixed CHO-K1 cells using a commercially availableanti-myc antibody (Cell Signaling, MAB 2276).

b. Immunization

Balb/c mice were immunized with 25 μg of p3973 plasmid DNA together with4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man from PolyPlusTransfection) (150 mM PEI-Man in H₂O with 5% glucose) by intraperitonealinjection on days 0 and 14. On days 28 and 44 mice were immunizedsubcutaneously with KLH-conjugated peptides SEQ ID NO: 14 and SEQ ID NO:15 (100 μg each in PBS, JPT Peptide Technologies GmbH, Germany) togetherwith HPLC-purified PTO-CpG-ODN (25 μg in PBS; 5′-TCCATGACGTTCCTGACGTT;Eurofins MWG Operon, Germany). On days 64, 77 and 97 mice were immunizedby intraperitoneal injection with 2×10⁷ P3X63Ag8U.1 myeloma cellstransfected with p3953 vector to stably express CLDN6. Prior toadministration, cells were treated with mitomycin-C (2.5 μg/ml,Sigma-Aldrich, M4287). On days 64 and 97 cells were administeredtogether with HPLC-purified PTO-CpG-ODN (50 μg in PBS), on day 77together with incomplete Freund's adjuvant.

For generation of monoclonal antibodies, mice with detectable anti-CLDN6immune responses were boosted four days prior to splenectomy byintraperitonal injection of 2×10⁷ HEK293 cells stably transfected withp3953 vector.

c. Testing of Monoclonal Antibodies Against CLDN6

Flow Cytometry

To test the binding of monoclonal antibodies to CLDN6 and its homologousHEK293T cells were transiently transfected with the correspondingclaudin-coding plasmid and the expression was analyzed by flowcytometry. In order to differentiate between transfected andnon-transfected cells, HEK293T cells were co-transfected with afluorescence marker as a reporter. 24 h post transfection cells wereharvested with 0.05% trypsin/EDTA, washed with FACS buffer (PBScontaining 2% FCS and 0.1% sodium azide) and resuspended in FACS bufferat a concentration of 2×10⁶ cells/ml. 100 μl of the cell suspension wereincubated with the appropriate antibody at indicated concentrations for30 min at 4° C. A cross-reactive antibody was used to detect CLDN6 andCLDN9 expression. The commercially available mouse anti-claudinantibodies anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219)served as positive controls, whereas mouse IgG2a (Sigma, M9144) andIgG2b (Sigma, M8894), respectively, served as isotype control. The cellswere washed three times with FACS buffer and incubated with anAPC-conjugated anti-mouse IgG 1+2a+2b+3a specific secondary antibody(Dianova, 115-135-164) for 30 min at 4° C. The cells were washed twiceand resuspended in FACS buffer. The binding was analyzed by flowcytometry using a BD FACSArray. The expression of the fluorescencemarker was plotted on the horizontal axis against the antibody bindingon the vertical axis.

CDC

The complement dependent cytotoxicity (CDC) was determined by measuringthe content of intracellular ATP in non-lysed cells after the additionof human complement to the target cells incubated with anti-CLDN6antibodies. As a very sensitive analytical method the luminescentreaction of luciferase was used for measuring ATP.

CHO-K1 cells stably transfected with CLDN6 (CHO-K1-CLDN6) were harvestedwith 0.05% trypsin/EDTA, washed twice with X-Vivo 15 medium (Lonza,BE04-418Q) and suspended at a concentration of 1×10⁷ cells/ml in X-Vivo15 medium. 250 μl of the cell suspension were transferred into a 0.4 cmelectroporation cuvette and mixed with 7 μg of in vitro transcribed RNAencoding for luciferase (luciferase IVT RNA). The cells wereelectroporated at 200 V and 300 μF using a Gene Pulser Xcell (Bio Rad).After electroporation, the cells were suspended in 2.4 ml pre-warmedD-MEM/F12 (1:1) with GlutaMax-I medium (Invitrogen, 31331-093)containing 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin and 1.5 mg/mlG418. 50 μl of the cell suspension per well were seeded into a white96-well PP-plate and incubated at 37° C. and 7.5% CO₂. 24 h postelectroporation 50 μl monoclonal murine anti-CLDN6 antibodies in 60%RPMI (containing 20 mM HEPES) and 40% human serum (serum pool obtainedfrom six healthy donors) were added to the cells at indicatedconcentrations. 10 μl 8% (v/v) Triton X-100 in PBS per well were addedto total lysis controls, whereas 10 μl PBS per well were added to maxviable cells controls and to the actual samples. After an incubation of80 min at 37° C. and 7.5% CO₂ 50 μl luciferin mix (3.84 mg/mlD-luciferin, 0.64 U/ml ATPase and 160 mM HEPES in ddH₂O) were added perwell. The plate was incubated in the dark for 45 min at RT. Theluminescence was measured using a luminometer (Infinite M200, TECAN).Results are given as integrated digital relative light units (RLU).

NEC8 cells were electroporated at 200 V and 400 μF and cultivated inRPMI 1640 with GlutaMAX-I medium (Invitrogen, 61870) containing 10%(v/v) FCS.

The specific lysis is calculated as:

${{specific}\mspace{14mu}{{lysis}\mspace{14mu}\lbrack\%\rbrack}} = {100 - \left\lbrack {\frac{\left( {{sample} - {{total}\mspace{14mu}{lysis}}} \right)}{\left( {{\max\mspace{14mu}{viable}\mspace{14mu}{cells}} - {{total}\mspace{14mu}{lysis}}} \right)} \times 100} \right\rbrack}$

-   -   max viable cells: 10 μl PBS, without antibody    -   total lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without        antibody        Early Treatment

For early antibody treatments 2×10⁷ NEC8 cells in 200 μl PBS weresubcutaneously inoculated into the flank of athymic Nude-Foxn1^(nu)mice. Each experimental group consisted of ten 6-8 week-old female mice.Three days after inoculation 200 μg of purified murine monoclonalantibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A were applied for46 days by alternating intravenous and intraperitoneal injections twicea week. Experimental groups treated with PBS served as a negativecontrols. The tumor volume (TV=(length×width²)/2) was monitoredbi-weekly. TV is expressed in mm³, allowing construction of tumor growthcurves over time. When the tumor reached a volume greater than 1500 mm³mice were killed.

d. Results

Murine monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67Ashowed strong binding to human CLDN6 and the CLDN6 SNP (singlenucleotide polymorphism) variant I143V while no binding to CLDN3, 4, and9 was observed (FIG. 6).

MuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A exhibited very low EC50values (EC50 200-500 ng/ml) and saturation of binding was achieved atlow concentrations (FIG. 7).

MuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A exhibited dose-dependent CDCactivity and induced CDC at low concentrations (FIG. 8). The anti-CLDN6antibodies muMAB 65A and 66B induced CDC on NEC8 cells in a dosedependent manner (FIG. 9). Target specificity of muMAB 65A and 66B wasproved by using NEC8 LVTS2 54 cells (CLDN6 knock-down).

Furthermore, muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A showed tumorgrowth inhibition in mice engrafted with NEC8 cells (FIG. 10).

Example 7 Generation and Testing of Chimeric Monoclonal AntibodiesAgainst CLDN6

a. Generation of Mouse/Human Chimeric Monoclonal Antibodies

For chimerization, the murine heavy chain and light chain variableregion including leader sequences were amplified by PCR using primerslisted in the table below. The murine heavy chains were fused by an ApaIrestriction site (5′-GGGCCC-3′) to the N-terminal part of the humanFcgamma1 chain, which was encoded by the expression vector. Variabledomains of the murine kappa chain including leader sequences were clonedin front of the constant region using a BsiWI restriction site. Thecorrect orientation of the constant region in the vector, i.e. suitablefor the preceeding promoter of the vector, was verified by sequencing.Due to the position of the ApaI restriction site, any amplification of avariable region including leader sequence for this purpose has toinclude the first 11 nucleotides of the sequence of the human gamma-1constant region in addition to the sequence of the ApaI site. Thenucleotide sequence of human gamma-1 heavy chain constant region islisted as SEQ ID NO: 24, the amino acid sequence of the thus expressedhuman gamma-1 constant region is listed as SEQ ID NO: 25. The nucleotidesequence encoding the constant part of the kappa light chain is listedas SEQ ID NO: 26, the respective amino acid sequence is listed as SEQ IDNO: 27.

TABLE 1 Mouse hybridoma cell lines used for antibody cloning PrimermuMAB Isotype SEQ ID NOs: heavy chain 64A IgG2a 17, 18 89A IgG2a 17, 1961D IgG2a 17, 20 67A IgG2a 17, 20 light chain 64A IgK 21, 22 89A IgK 21,23 61D IgK 21, 22 67A IgK 21, 22

Corresponding to their murine counterparts the chimeric monoclonalantibodies were named adding the prefix “chim”, e.g. chimAB 64A.

Amplification of the murine variable regions of light and heavy chainsincluding leader sequences was carried out according to the “step-outPCR” method described in Matz et al. (Nucleic Acids Research, 1999, Vol.27, No. 6). For this, total RNA was prepared from monoclonal hybridomacell lines (see Tab. 1) by standard methods known to those skilled inthe art, for example with the use of RNeasy Mini Kit (Qiagen). Singlestranded cDNA was prepared according to the “template-switch” methodalso described in Matz et al. (Nucleic Acids Research, 1999, Vol. 27,No. 6, 1558). In addition to a (dT)30 oligomer (SEQ ID NO: 28), itincluded a DNA/RNA hybrid oligomer (SEQ ID NO: 29) serving as an 5′adaptor for template switching during polymerization of the cDNA strand.In this adaptor oligomer the last three nucleotides were ribo-instead ofdeoxyribonucleotides. The subsequent “step-out PCR” used an antisenseoligomer targeted to the constant region of the mouse kappa chain or tothe constant region of the subclass 2a of the gamma chain (SEQ ID NO: 30and 31, respectively). The IgG subclass of the murine monoclonalantibody produced by the hybridoma cell lines was afore immunologicallyanalyzed with IsoStrip (Roche), and the appropriate antisense oligomerwas chosen accordingly (see Tab. 1). A primer mix served as the senseoligomer in the “step-out PCR”, comprising the two oligomers listed inSEQ ID NO: 32 and 33.

The identified murine variable regions including leader sequences werethen amplified by PCR omitting the 5′ UTR and the 3′ mouse constantregion, adding restriction sites to the ends which allowed subcloninginto the prepared expression vectors carrying the human constantregions. In addition, the sense oligomers provided a consensus Kozaksequence (5′-GCCGCCACC-3′) and the antisense oligomers for heavy chainvariable regions included the first 11 nucleotides of the human gamma-1constant region in addition to the ApaI restriction site (see Tab. 1,SEQ ID NOs: 17 to 23). Kappa light chain variable regions includingleader sequences were cloned using HindIII and BsiWI restrictionenzymes, gamma heavy chain variable regions demanded HindIII and ApaIrestriction enzymes.

Further murine variable regions of light and heavy chains includingleader sequences were amplified and further chimeric monoclonalantibodies against CLDN6 generated in accordance to the protocoldisclosed above.

b. Production of Chimeric Monoclonal Anti-CLDN6 Antibodies

Chimeric monoclonal antibodies were transiently expressed in HEK293Tcells (ATCC CRL-11268) transfected with plasmid DNA coding for the lightand heavy chains of the corresponding antibody. 24 h before transfection8×10⁷ cells were seeded on 145 mm cell culture plates and cultivated in25 ml HEK293T-medium (DMEM/F12+GlutaMAX-I, 10% FCS, 1%penicillin/streptomycin). 20 μg plasmid DNA were dissolved in 5 mlHEK293T-medium without supplements per cell culture plate. After adding75 μl linear polyethylenimine (PEI) (1 mg/ml) (Polyscience, 23966) the(DNA:PEI)-mixture was incubated 15 min at RT. Thereafter, thetransfection-mix was added dropwise to the cells. 24 h post transfectionthe HEK293T-medium was replaced with Pro293a-medium (Lonza, BE12-764Q)containing 1% penicillin/streptomycin. For optimal expression, thetransfected cells were cultivated at 37° C. and 7.5% CO₂ for additional96 to 120 h. The supernatant was harvested and the chimeric antibody waspurified by FPLC using protein-A columns. The concentration of theantibody was determined and quality was tested by SDS-PAGE.

c. Testing of Chimeric Monoclonal Antibodies Against CLDN6

Flow Cytometry

To test the specificities and affinities of CLDN6-specific chimericmonoclonal antibodies binding to HEK293 cells stably transfected withCLDN3, 4, 6 or 9, respectively, and tumor cell lines that endogenouslyexpress CLDN6 was analyzed by flow cytometry. Therefore, cells wereharvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBScontaining 2% FCS and 0.1% sodium azide) and resuspended in FACS bufferat a concentration of 2×10⁶ cells/ml. 100 μl of the cell suspension wereincubated with the appropriate antibody at indicated concentrations for60 min at 4° C. A chimeric cross-reactive antibody (chimAB 5F2D2) wasused to detect CLDN6 and CLDN9 expression. The commercially availablemouse anti-claudin antibodies anti-CLDN3 (R&D, MAB4620) and anti-CLDN4(R&D, MAB4219) served as positive controls, whereas human IgG 1-kappa(Sigma, I5154) served as a negative control. The cells were washed threetimes with FACS buffer and incubated for 30 min at 4° C. with anAPC-conjugated goat anti-human IgG Fc-gamma (Dianova, 109-136-170) or anAPC-conjugated anti-mouse IgG 1+2a+2b+3a (Dianova, 115-135-164) specificsecondary antibody, respectively. The cells were washed twice andresuspended in FACS buffer. The binding was analyzed by flow cytometryusing a BD FACSArray.

CDC

The complement dependent cytotoxicity (CDC) was determined by measuringthe content of intracellular ATP in non-lysed cells after the additionof human complement to the target cells incubated with anti-CLDN6antibodies. As a very sensitive analytical method the bioluminescentreaction of luciferase is used for measuring ATP.

In this assay, NEC8 wildtype cells (CLDN6 positive) and NEC8 CLDN6knock-down cells (CLDN6 negative) were used which both were stablytransduced with luciferase expression construct. The cells wereharvested with 0.05% Trypsin/EDTA and adjusted to a concentration of2×10⁵ cells/ml in RPMI with GlutaMax-I medium (Invitrogen, 61870-010)containing 10% (v/v) FCS. 1×10⁴ cells were seeded into a white 96-wellPP-plate and incubated for 24 h at 37° C. and 5% CO₂. After incubation,50 μl monoclonal chimeric anti-CLDN6 antibodies in 60% RPMI (containing20 mM HEPES) and 40% human serum (serum pool obtained from six healthydonors) were added to the cells at indicated concentrations. 10 μl 8%(v/v) Triton X-100 in PBS per well were added to total lysis controls,whereas 10 μl PBS per well were added to max viable cells controls andto the actual samples. After a further incubation of 80 min at 37° C.and 5% CO₂ 50 μl luciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPaseand 160 mM HEPES in ddH₂O) was added per well. The plate was incubatedin the dark at RT for 45 min. The bioluminescence was measured using aluminometer (Infinite M200, TECAN). Results are given as integrateddigital relative light units (RLU).

The specific lysis is calculated as:

${{specific}\mspace{14mu}{{lysis}\mspace{14mu}\lbrack\%\rbrack}} = {100 - \left\lbrack {\frac{\left( {{sample} - {{total}\mspace{14mu}{lysis}}} \right)}{\left( {{\max\mspace{14mu}{viable}\mspace{14mu}{cells}} - {{total}\mspace{14mu}{lysis}}} \right)} \times 100} \right\rbrack}$

-   -   max viable cells: 10 μl PBS, without antibody    -   total lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without        antibody        ADCC

The antibody dependent cellular cytotoxicity (ADCC) was determined bymeasuring the content of intracellular ATP in non-lysed cells after theaddition of human PBMC to the target cells incubated with anti-CLDN6antibodies. As a very sensitive analytical method the bioluminescentreaction of luciferase is used for measuring ATP.

In this assay, NEC-8 wildtype cells (CLDN6 positive) and NEC-8 CLDN6knock-down cells (CLDN6 negative) were used which both were stablytransduced with luciferase expression construct. The cells wereharvested with 0.05% Trypsin/EDTA and adjusted to a concentration of2×10⁵ cells/ml in RPMI with GlutaMax-I medium (Invitrogen, 61870-010)containing 10% (v/v) FCS and 20 mM Hepes. 1×10⁴ cells were seeded into awhite 96-well PP-plate and incubated 4 h at 37° C. and 5% CO₂.

PBMC were isolated from human donor blood samples by density gradientcentrifugation using Ficoll Hypaque (GE Healthcare, 17144003). The PMBCcontaining interphase was isolated and cells were washed twice withPBS/EDTA (2 mM). 1×10⁸ PBMC were seeded in 50 ml X-Vivo 15 medium(Lonza, BE04-418Q) containing 5% heat-inactivated human serum (Lonza,US14-402E) and incubated for 2 h at 37° C. and 5% CO₂.

4 h post seeding of the target cells (NEC-8) 25 μl monoclonal chimericanti-CLDN6 antibodies in PBS were added to the cells at indicatedconcentrations. Nonadherent PBMC, which separated within the 2 hincubation from adherent monocytes, were harvested and adjusted to 8×10⁶cells/ml in X-vivo 15 medium. 25 μl of this cell suspension was added tothe target cells and the monoclonal chimeric anti-CLDN6 antibodies. Theplates were incubated for 24 h at 37° C. and 5% CO₂.

After the 24 h incubation 10 μl 8% (v/v) Triton X-100 in PBS per wellwere added to total lysis controls, whereas 10 μl PBS per well wereadded to max viable cells controls and to the actual samples. 50 μlluciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPase and 160 mM HEPESin ddH₂O) was added per well. The plate was incubated in the dark at RTfor 30 min. The bioluminescence was measured using a luminometer(Infinite M200, TECAN). Results are given as integrated digital relativelight units (RLU).

The specific lysis is calculated as:

${{specific}\mspace{14mu}{{lysis}\mspace{14mu}\lbrack\%\rbrack}} = {100 - \left\lbrack {\frac{\left( {{sample} - {{total}\mspace{14mu}{lysis}}} \right)}{\left( {{\max\mspace{14mu}{viable}\mspace{14mu}{cells}} - {{total}\mspace{14mu}{lysis}}} \right)} \times 100} \right\rbrack}$

-   -   max viable cells: 10 μl PBS, without antibody    -   total lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without        antibody        d. Results

Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89Ashowed strong binding to human CLDN6 while no binding to CLDN3, 4, and 9was observed (FIG. 11).

Regarding binding to human CLDN6 stably expressed on the surface ofHEK293 cells, anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and89A exhibit very low EC50 values (EC50 450-600 ng/ml) and saturation ofbinding was achieved at low concentrations. ChimAB 67A and 61D showedlow (EC50 1000 ng/ml) and medium (EC50 2300 ng/ml) EC50 values,respectively (FIG. 12).

Regarding binding to CLDN6 endogenously expressed in NEC8 cells,anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and 89A exhibitedvery low EC50 values (EC50 600-650 ng/ml) and saturation of binding wasachieved at low concentrations, whereas chimAB 61D and 67A showed medium(EC50 1700 ng/ml) and high (EC50 6100 ng/ml) EC50 values, respectively(FIG. 13).

Regarding binding to CLDN6 endogenously expressed in OV90 cells,anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and 89A exhibitedvery low EC50 values (EC50 550-600 ng/ml) and saturation of binding wasachieved at low concentrations. ChimAB 61D and 67A showed medium EC50values (EC50 1500 ng/ml and EC50 2300 ng/ml, respectively) (FIG. 14).

Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89Aexhibited CDC activity in a dose-dependent manner on NEC-8 cells (FIG.15).

Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89Aexhibited dose-dependent ADCC activity on NEC-8 cells and induced ADCCeven at low antibody concentrations (FIG. 16).

These results clearly show the specificity of these chimeric monoclonalantibodies for CLDN6.

Example 8 Treatment Using Monoclonal Antibodies Against CLDN6

Early Treatment

For early antibody treatments 2×10⁷ NEC8 cells in 200 μl RPMI medium(Gibco) were subcutaneously inoculated into the flank of athymicNude-Foxn1^(nu) mice. Each experimental group consisted of ten 6-8week-old female mice. Three days after tumor cell inoculation 200 μg ofpurified murine monoclonal antibody muMAB 89A was applied for sevenweeks by alternating intravenous and intraperitoneal injections twice aweek. Experimental group treated with PBS served as negative control.The tumor volume (TV=(length×width²)/2) was monitored bi-weekly. TV isexpressed in mm³, allowing construction of tumor growth curves overtime. When the tumors reached a volume greater than 1500 mm³ mm micewere sacrificed.

Advanced Treatments

For antibody treatments of advanced xenograft tumors 2×10⁷ NEC8 cells in200 μl RPMI medium (Gibco) were subcutaneously inoculated into the flankof 6-8 week-old female athymic Nude-Foxn1^(nu) mice. The tumor volume(TV=(length×width²)/2) was monitored bi-weekly. TV is expressed in mm³,allowing construction of tumor growth curves over time. 15 to 17 daysafter tumor cell inoculation mice were divided into treatment groups ofeight animals per cohorte with homogenous tumor sizes of above 80 mm³.200 μg of purified murine monoclonal antibodies muMAB 61D, 64A, 67A and89A were applied for five weeks by alternating intravenous andintraperitoneal injections twice a week. Experimental groups treatedwith PBS and an unspecific antibody served as negative controls. Whenthe tumors reached a volume bigger than 1500 mm³ mice were sacrificed.

In an early treatment xenograft model using mice engrafted with thetumor cell line NEC8, mice treated with murine monoclonal antibodiesmuMAB 61D, 64A and 67A did not show any tumor growth even after stoppingthe immunotherapy (FIG. 17).

In an early treatment xenograft model using mice engrafted with thetumor cell line NEC8, muMAB 89A showed tumor growth inhibition and notumors were detectable in mice treated with muMAB89A at the end of thestudy (FIG. 18).

In an advanced treatment xenograft model using mice engrafted with thetumor cell line NEC8, muMAB 64A showed an inhibition of tumor growth(FIG. 19).

In an advanced treatment xenograft model using mice engrafted with thetumor cell line NEC8, mice treated with muMAB 64A showed prolongedsurvival (FIG. 20).

In an advanced treatment xenograft model using mice engrafted with thetumor cell line NEC8, inhibition of tumor growth was achieved with themurine monoclonal anti-CLDN6 antibodies muMAB 61D, 67A and 89A (FIG.21).

In an advanced treatment xenograft model using mice engrafted with thetumor cell line NEC8, mice treated with the CLDN6 specific antibodymuMAB 61D or 67A showed prolonged survival (FIG. 22).

In an advanced treatment xenograft model using mice engrafted with NEC8wildtype and NEC8 cells with a stable CLDN6 knock-down, muMAB 64A and89A only show a therapeutic effect in mice engrafted with NEC8 wildtypebut not in mice engrafted with NEC8 CLDN6 knock-down cells demonstratingCLDN6-specificity of the antibody in vivo (FIG. 23).

Example 9 High-Resolution Eitope Mapping of Monoclonal AntibodiesAgainst CLDN6

The CLDN6 specific monoclonal antibodies only show very weak (if any)binding to linear peptides in ELISA epitope-mapping studies, implyingthat their epitopes are conformational. To analyze the interactionbetween antibodies described herein and CLDN6 in its native conformationsite-directed mutagenesis in mammalian cell culture was used as anepitope-mapping technique. Alanine scanning mutagenesis of amino acids27-81 and 137-161 within the first and second extracellular domain,respectively, was performed. Following transient expression in HEK293Tcells, CLDN6 mutants were assessed for their ability to be bound byspecific monoclonal antibodies. Impaired binding of a specificmonoclonal antibody to a CLDN6 mutant suggest that the mutated aminoacid is an important contact and/or conformational residue. The bindingwas analyzed by flow cytometry. To discriminate between transfected andnon-transfected cell populations, cells were co-transfected with afluorescence marker.

The amino acid residues of CLDN6 that are important for the interactionwith CLDN6 specific chimeric antibodies have been systematicallyidentified by alanine-scanning. Alanine and glycine mutations weregenerated by site-directed mutagenesis (GENEART AG, Germany). To testthe binding of monoclonal chimeric antibodies to wild-type CLDN6 and itsmutants HEK293T cells were transiently transfected with thecorresponding claudin-coding plasmid and the expression was analyzed byflow cytometry. In order to differentiate between transfected andnon-transfected cells, HEK293T cells were co-transfected with afluorescence marker as a reporter. 24 h post transfection cells wereharvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBScontaining 2% FCS and 0.1% sodium azide) and resuspended in FACS bufferat a concentration of 2×10⁶ cells/ml. 100 μl of the cell suspension wereincubated with 10 μg/ml antibody for 45 min at 4° C. The commerciallyavailable mouse anti-CLDN6 (R&D, MAB3656) was used as a control todetect cell-surface expression of CLDN6 mutants. The cells were washedthree times with FACS buffer and incubated with an APC-conjugated goatanti-human IgG Fc-gamma (Dianova, 109-136-170) or an APC-conjugatedanti-mouse IgG 1+2a+2b+3a specific secondary antibody (Dianova,115-135-164) for 30 min at 4° C. The cells were washed twice andresuspended in FACS buffer. The binding within the transfected cellpopulation was analyzed by flow cytometry using a BD FACSArray.Therefore, the expression of the fluorescence marker was plotted on thehorizontal axis against the antibody binding on the vertical axis. Theaverage signal intensity of a monoclonal chimeric CLDN6 specificantibody bound to mutant CLDN6 was expressed as the percentage ofwild-type binding. Amino acids that are essential for binding showed nobinding after being mutated whereas amino acids that support bindingonly showed reduced binding compared to wild-type.

High resolution epitope-mapping demonstrated that the amino acids F35,G37, S39 and possibly T33 of the first extracellular domain of CLDN6 areimportant for the interaction with the CLDN6 specific chimericantibodies chimAB 61D, 64A, 67A and 89A. Residue 140 is essential forthe binding of chimAB 89A and it contributes to the binding of chimAB61D and 67A. In addition, L151 of the second extracellular domain ofCLDN6 is important for the interaction with chimAB 67A (FIG. 24).

The invention claimed is:
 1. A monoclonal antibody which is capable ofbinding to CLDN6, wherein the antibody comprises a set of heavy chainCDR (HCDR) and light chain CDR (LCDR) selected from the group consistingof (a) a HCDR1 of amino acid sequence GYSFTGYT (SEQ ID NO: 47)identified within SEQ ID NO: 34, a HCDR2 of amino acid sequence INPYNGGT(SEQ ID NO: 54) identified within SEQ ID NO: 34, a HCDR3 of amino acidsequence ARDYGYVLDY (SEQ ID NO: 55) identified within SEQ ID NO: 34, aLCDR1 of amino acid sequence SSVSY (SEQ ID NO: 56) identified within SEQID NO: 35, a LCDR2 of amino acid sequence STS (SEQ ID NO: 53) identifiedwithin SEQ ID NO: 35, and a LCDR3 of amino acid sequence QQRSIYPPWT (SEQID NO: 57) identified within SEQ ID NO: 35; (b) a HCDR1 of amino acidsequence GYSFTGYT (SEQ ID NO: 47) identified within SEQ ID NO: 36, aHCDR2 of amino acid sequence INPYNGGT (SEQ ID NO: 54) identified withinSEQ ID NO: 36, a HCDR3 of amino acid sequence ARDYGFVLDY (SEQ ID NO: 58)identified within SEQ ID NO: 36, a LCDR1 of amino acid sequence SSVSY(SEQ ID NO: 56) identified within SEQ ID NO: 37, a LCDR2 of amino acidsequence STS (SEQ ID NO: 53) identified within SEQ ID NO: 37, and aLCDR3 of amino acid sequence QQRSNYPPWT (SEQ ID NO: 59) identifiedwithin SEQ ID NO: 37; (c) a HCDR1 of amino acid sequence GYSFTGYT (SEQID NO: 47) identified within SEQ ID NO: 38, a HCDR2 of amino acidsequence INPYNGGI (SEQ ID NO: 60) identified within SEQ ID NO: 38, aHCDR3 of amino acid sequence ARDFGYVLDY (SEQ ID NO: 61) identifiedwithin SEQ ID NO: 38, a LCDR1 of amino acid sequence SSVSY (SEQ ID NO:56) identified within SEQ ID NO: 39, a LCDR2 of amino acid sequence STS(SEQ ID NO: 53) identified within SEQ ID NO: 39, and a LCDR3 of aminoacid sequence QQRSTYPPWT (SEQ ID NO: 62) identified within SEQ ID NO:39; and (d) a HCDR1 of amino acid sequence GYSFTGYT (SEQ ID NO: 47)identified within SEQ ID NO: 40, a HCDR2 of amino acid sequence INPYNGGS(SEQ ID NO: 63) identified within SEQ ID NO: 40, a HCDR3 of amino acidsequence ARDYGYVFDY (SEQ ID NO: 64) identified within SEQ ID NO: 40, aLCDR1 of amino acid sequence SSVNY (SEQ ID NO: 65) identified within SEQID NO: 41, a LCDR2 of amino acid sequence STS (SEQ ID NO: 53) identifiedwithin SEQ ID NO: 41, and a LCDR3 of amino acid sequence QQRNNYPPWT (SEQID NO: 66) identified within SEQ ID NO:
 41. 2. The antibody of claim 1,which is not capable of detectably binding to (i) CLDN9 associated withthe surface of a cell that expresses CLDN9 and (ii) CLDN4 associatedwith the surface of a cell that expresses CLDN4.
 3. The antibody ofclaim 1, which is not capable of detectably binding to CLDN3 associatedwith the surface of a cell that expresses CLDN3.
 4. The antibody of orclaim 1, which is specific for CLDN6.
 5. The antibody of or claim 1,wherein CLDN6 has the amino acid sequence of SEQ ID NO: 2 or the aminoacid sequence of SEQ ID NO:
 8. 6. The antibody of claim 1, which (a) hasone or more of the following activities selected from the groupconsisting of: (i) killing of a cell expressing CLDN6, (ii) inhibitionof proliferation of a cell expressing CLDN6, (iii) inhibition of colonyformation of a cell expressing CLDN6, (iv) mediating remission ofestablished tumors, (v) preventing formation or re-formation of tumors,and (vi) inhibition of metastasis of a cell expressing CLDN6; (b)exhibits one or more immune effector functions against a cell carryingCLDN6 in its native conformation, wherein the one or more immuneeffector functions are optionally selected from the group consisting ofcomplement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), induction of apoptosis, andinhibition of proliferation; or (c) has one or more of the activities of(a) and exhibits (b).
 7. The antibody of claim 1, which is a chimeric orhumanized antibody, or a fragment of an antibody.
 8. An antibodyselected from the group consisting of (i) an antibody produced by orobtainable from a clone deposited under the accession no. DSM ACC3067(GT512muMAB 59A), DSM ACC3068 (GT512muMAB 60A), DSM ACC3069 (GT512muMAB61D), DSM ACC3070 (GT512muMAB 64A), DSM ACC3071 (GT512muMAB 65A), DSMACC3072 (GT512muMAB 66B), DSM ACC3073 (GT512muMAB 67A), DSM ACC3089(GT512muMAB 55A), or DSM ACC3090 (GT512muMAB 89A), (ii) an antibodywhich is a chimerized or humanized form of the antibody under (i), and(iii) an antibody comprising the antigen binding portion or antigenbinding site of the antibody under (i).
 9. A hybridoma capable ofproducing the antibody of claim 1 or claim
 8. 10. A hybridoma depositedunder the accession no. DSM ACC3067 (GT512muMAB 59A), DSM ACC3068(GT512muMAB 60A), DSM ACC3069 (GT512muMAB 61D), DSM ACC3070 (GT512muMAB64A), DSM ACC3071 (GT512muMAB 65A), DSM ACC3072 (GT512muMAB 66B), DSMACC3073 (GT512muMAB 67A), DSM ACC3089 (GT512muMAB 55A), or DSM ACC3090(GT512muMAB 89A).
 11. A conjugate comprising an antibody of claim 1,coupled to a therapeutic agent, wherein the therapeutic agent isoptionally a toxin, a radioisotope, a drug or a cytotoxic agent.
 12. Apharmaceutical composition comprising (i) the antibody of claim 1; (ii)a conjugate comprising an antibody of claim 1, coupled to a therapeuticagent, wherein the therapeutic agent is optionally a toxin, aradioisotope, a drug or a cytotoxic agent, and a pharmaceuticallyacceptable carrier; or (i) and (ii).
 13. A method of inhibiting growthof a cell expressing CLDN6 and being characterized by association ofCLDN6 with its cell surface, comprising contacting the cell with (i) anantibody of claim 1; (ii) a conjugate comprising an antibody of claim 1,coupled to a therapeutic agent, wherein the therapeutic agent isoptionally a toxin, a radioisotope, a drug or a cytotoxic agent; or (i)and (ii).
 14. A method of killing a cell expressing CLDN6 and beingcharacterized by association of CLDN6 with its cell surface, comprisingcontacting the cell with (i) an antibody of claim 1; (ii) a conjugatecomprising an antibody of claim 1, coupled to a therapeutic agent,wherein the therapeutic agent is optionally a toxin, a radioisotope, adrug or a cytotoxic agent; or (i) and (ii).
 15. A method of treating orpreventing a disease or disorder involving a cell expressing CLDN6 andbeing characterized by association of CLDN6 with its cell surface in asubject, comprising administering to said subject (a) an antibody ofclaim 1; (b) a conjugate comprising an antibody of claim 1, coupled to atherapeutic agent, wherein the therapeutic agent is optionally a toxin,a radioisotope, a drug or a cytotoxic agent; or (c) a pharmaceuticalcomposition comprising (i) the antibody of claim 1; (ii) a conjugatecomprising an antibody of claim 1, coupled to a therapeutic agent,wherein the therapeutic agent is optionally a toxin, a radioisotope, adrug or a cytotoxic agent, and a pharmaceutically acceptable carrier; or(i) and (ii).
 16. The method of claim 15, wherein the disease ordisorder is a tumor-related disease, wherein the tumor-related diseaseis optionally a cancer selected from the group consisting of ovariancancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer, agerm cell tumor such as a teratocarcinoma or an embryonal carcinoma, inparticular a germ cell tumor of the testis, and the metastatic formsthereof.
 17. A method of inhibiting metastatic spread of a cellexpressing CLDN6 and being characterized by association of CLDN6 withits cell surface, comprising contacting the cell with (i) an antibody ofclaim 1; (ii) a conjugate comprising an antibody of claim 1, coupled toa therapeutic agent, wherein the therapeutic agent is optionally atoxin, a radioisotope, a drug or a cytotoxic agent; or (i) and (ii). 18.The antibody of claim 1, wherein said antibody induces CDC- orADCC-mediated killing of cells expressing CLDN6.
 19. The antibody ofclaim 18, wherein said antibody does not detectably bind to CLDN9. 20.The monoclonal antibody of claim 1, wherein the antibody comprises a setof antibody heavy chain sequence and an antibody light chain sequenceselected from the group consisting of (a) an antibody heavy chainsequence comprising SEQ ID NO: 34, and an antibody light chain sequencecomprising SEQ ID NO: 35; (b) an antibody heavy chain sequencecomprising SEQ ID NOs: 36, and an antibody light chain sequencecomprising SEQ ID NO: 37; (c) an antibody heavy chain sequencecomprising SEQ ID NOs: 38, and an antibody light chain sequencecomprising SEQ ID NO: 39; and (d) an antibody heavy chain sequencecomprising SEQ ID NO: 40 and an antibody light chain sequence comprisingSEQ ID NO: 41.